Novel protein, dna thereof and process for producing the same

ABSTRACT

The present invention relates to a novel protein containing a peptide having a ligand activity to a G-protein copuled receptor protein; amides, esters, salts of the peptides; and drugs containing the same.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a protein comprising a peptide whichhas a ligand activity to a novel G protein-coupled receptor proteinderived from rat brain stem and its peripheral region and human brain,and a amide or ester of the peptide, or a salt thereof.

BACKGROUND ART

Physiological active substances such as various hormones andneurotransmitters regulate the biological function via specific receptorproteins present on cell membranes. Many of these receptor proteins arecoupled with guanine nucleotide-binding protein (hereinafter sometimessimply referred to as G protein) and mediate the intracellular signaltransduction via activation of G protein. These receptor proteinspossess the common structure containing seven transmembrane domains andare thus collectively referred to as G protein-coupled receptors orseven-transmembrane receptors (7TMR).

G protein-coupled receptor proteins present on the cell surface of eachfunctional cell and organ in the body, and play important physiologicalroles as the target of the molecules that regulate the functions of thecells and organs, e.g., hormones, neurotransmitters, physiologicallyactive substances and the like. Receptors transmit signals to cells viabinding with physiologically active substances, and the signals inducevarious reactions such as activation and inhibition of the cells.

To clarify the relationship between substances that regulate complexbiological functions in various cells and organs, and their specificreceptor proteins, in particular, G protein-coupled receptor proteins,would elucidate the functional mechanisms in various cells and organs inthe body to provide a very important means for development of drugsclosely associated with the functions.

For example, in various organs, their physiological functions arecontrolled in vivo through regulation by many hormones, hormone-likesubstances, neurotransmitters or physiologically active substances. Inparticular, physiologically active substances are found in numeroussites of the body and regulate the physiological functions through theircorresponding receptor proteins. However, it is supposed that manyunknown hormones, neurotransmitters or many other physiologically activesubstances still exist in the body and, as to their receptor proteins,many of these proteins have not yet been reported. In addition, it isstill unknown if there are subtypes of known receptor proteins.

It is very important for development of drugs to clarify therelationship between substances that regulate elaborated functions invivo and their specific receptor proteins. Furthermore, for efficientscreening of agonists and antagonists to receptor proteins indevelopment of drugs, it is required to clarify functional mechanisms ofreceptor protein genes expressed in vivo and express the genes in anappropriate expression system.

In recent years, random analysis of cDNA sequences has been activelystudied as a means for analyzing genes expressed in vivo. The sequencesof cDNA fragments thus obtained have been registered on and published todatabases as Expressed Sequence Tag (EST). However, since many ESTscontain sequence information only, it is difficult to predict theirfunctions from the information.

Substances which inhibit the binding between G protein-coupled proteinsand physiologically active substances (i.e. ligands) and substanceswhich bind and induce signals similar to those induced byphysiologically active substances (i.e. ligands) have been used asantagonists and agonists specific to the receptors, or pharmaceuticalswhich regulate the biological functions. Therefore, discovering a novelG protein-coupled receptor which can be targeted for pharmaceuticaldevelopment, and a ligand specific to the receptor are very importantways for searching for an agonist and antagonist of the receptor.

Furthermore, elucidating the physiological function of receptors andpredicting the role of receptors in a human, which are carried out byobtaining a counterpart of a human gene encoding the receptor or ligandfrom a rodent (rat, mouse), chimpanzee, monkey, etc.; by investigatingthe chemical properties and biological activities of these geneproducts; and by examining precisely their qualitative and quantitativeprofiles inside an animal body or physiological mechanisms, are veryimportant for creating an effective pharmaceutical.

In addition, for selecting candidate compounds for an agonist orantagonist, considering species difference is an essential step for drugdevelopment.

SUMMARY OF THE INVENTION

The present invention provides, as described above, rat and mousehomologues of a human ligand protein specific to the human useful novelG-protein coupled receptor protein (hereinafter, which is referred to as“the ligand protein of the present invention”).

Until now, the inventors searched for a peptide which binds to theG-protein coupled receptor derived from rat brain stem and human brainand has activity of inducing increase in intracellular Ca ionconcentration. As a result, the inventors found that a C-terminalpeptide of a protein encoded by a tumor-metastasis suppressor gene,KiSS-1 (Genomics, vol. 54, 145-148, 1998) activated the receptor, andconfirmed that the peptide consisting of 54 amino acids of KiSS-1, and aC-terminal partial peptide thereof has the ligand activity (JP-A2000-312590).

It is expected that a peptide generated through cleavage of the KiSS-1gene product have activity of inhibiting tumor metastasis, because thegene is the tumor-metastasis suppressor gene. In addition, consideringthat the gene is expressed in a large amount in placenta, and that thehuman G-protein coupled receptor protein, hOT7T175 (in which h meanshuman) is expressed in large quantity in placenta, it is predicted thatthe peptide plays an important role in placenta. Further, becauseexpression of the receptor is also relatively high in pancreas inside ahuman body, the peptide may have some physiological function inpancreas. Rat ligand and rat receptor rOT7T175 (in which r means rat)are both highly expressed in cecum and large intestine, indicating somephysiological function of the peptide in these tissues. With extensiveresearch, the inventors successfully isolated cDNA encoding a sequencehighly homologous to the human KiSS-1 gene from rat liver cDNA using PCRprimers prepared on the basis of the sequence of human KiSS-1 gene, anddetermined its nucleic acid sequence completely. The amino acid sequencededuced from the nucleic acid sequence revealed that the nucleic acidsequence encodes a protein which has significantly high homology to theamino acid sequence of human KiSS-1. The inventors also isolated cDNAencoding a sequence highly homologous to the human KiSS-1 gene frommouse embryo cDNA, and confirmed that its sequence encodes a proteinwhich has significantly high homology to the amino acid sequence ofhuman and rat KiSS-1.

Based on these findings, the present inventors have continued furtherextensive studies and as a result, have come to accomplish the presentinvention. Thus, the present invention relates to the following:

-   -   (1) a protein comprising the same or substantially the same        amino acid sequence as that represented by SEQ ID NO:1 or NO:3,        or a salt thereof;    -   (2) the protein or salt thereof according to the above (1),        wherein substantially the same amino acid sequence is one        represented by SEQ ID NO:2;    -   (3) a partial peptide of the protein according to the above (1),        which comprises the amino acid sequence of from the 132 to 141th        residues from the N terminal of the amino acid sequence        represented by SEQ ID NO:1, or a salt thereof;    -   (4) the partial peptide or salt thereof according to the above        (3), which comprises the same or substantially the same amino        acid sequence as that of from the 127 to 141th residues from the        N terminal of the amino acid sequence represented by SEQ ID        NO:1;    -   (5) the partial peptide or salt thereof according to the above        (3), which has the same or substantially the same amino acid        sequence as that of from the 90 to 141th residues from the N        terminal of the amino acid sequence represented by SEQ ID NO:1;    -   (6) a partial peptide of the protein according to the above (1),        which has the same or substantially the same amino acid sequence        as that of from the 94 to 145th residues from the N terminal of        the amino acid sequence represented by SEQ ID NO:2, or a salt        thereof;    -   (7) a partial peptide of the protein according to the above (1),        which comprises the amino acid sequence of from the 110 to 119th        residues from the N terminal of the amino acid sequence        represented by SEQ ID NO:3, or a salt thereof;    -   (8) the partial peptide or salt thereof according to the above        (7), which comprises the same or substantially the same amino        acid sequence as that of from the 105 to 119th residues from the        N terminal of the amino acid sequence represented by SEQ ID        NO:3;    -   (9) the partial peptide or salt thereof according to the above        (7), which has the same or substantially the same amino acid        sequence as that of from the 68 to 119th residues from the N        terminal of the amino acid sequence represented by SEQ ID NO:3;    -   (10) a polynucleotide comprising a polynucleotide encoding the        protein according to the above (1);    -   (11) a polynucleotide comprising a polynucleotide encoding the        partial peptide according to the above (3), (6) or (7);    -   (12) the polynucleotide according to the above (10) or (11),        which is DNA;    -   (13) the polynucleotide according to the above (10), which has        the nucleic acid sequence represented by SEQ ID NO:4, NO:5 or        NO:6;    -   (14) a recombinant vector comprising the polynucleotide        according to the above (10) or (11);    -   (15) a transformant transformed with the recombinant vector        according to the above (14);    -   (16) a method of producing the protein or salt thereof according        to the above (1), or the partial peptide or salt thereof        according to the above (3), (6) or (7), which comprises        culturing the transformant according to (15), and making it        produce and accumulate the protein according to the above (1),        or the partial peptide according to the above (3), (6) or (7);    -   (17) an antibody to the protein or salt thereof according to the        above (1), or the partial peptide or salt thereof according to        the above (3), (6) or (7);    -   (18) the antibody according to (17), which is a neutralizing        antibody capable of inactivating signal transduction of the        protein according to (1) or the partial peptide according to the        above (3), (6) or (7);    -   (19) a method of screening a compound or salt thereof which        alters the binding property between a receptor and the protein        or salt thereof according to the above (1) or the partial        peptide or salt thereof according to the above (3), (6) or (7),        which comprises using the protein or salt thereof according to        the above (1) or the partial peptide or salt thereof according        to the above (3), (6) or (7);    -   (20) a kit for screening a compound or salt thereof which alters        the binding property between a receptor and the protein or salt        thereof according to the above (1) or the partial peptide or        salt thereof according to the above (3), (6) or (7), which        comprises the protein or salt thereof according to the above (1)        or the partial peptide or salt thereof according to the above        (3), (6) or (7);    -   (21) the screening method according to the above (19) or the        screening kit according to the above (20), wherein the receptor        is a protein or salt thereof having the same or substantially        the same amino acid sequence as that represented by SEQ ID NO:7,        NO:8 or NO:24;    -   (22) a compound or salt thereof which alters the binding        property between a receptor and the protein or salt thereof        according to any one of the above (1) and (3) to (5), which is        obtainable using the screening method according to the        above (19) or the screening kit according to the above (20);    -   (23) the compound or salt thereof according to the above (22),        which is an agonist;    -   (24) a pharmaceutical composition comprising a compound or salt        thereof which alters the binding property between a receptor and        the protein or salt thereof according to the above (1) or the        partial peptide or salt thereof according to the above (3), (6)        or (7), which is obtainable using the screening method according        to the above (19) or the screening kit according to the above        (20);    -   (25) the pharmaceutical composition according to the above (24),        which is an agent for inhibiting tumor metastasis;    -   (26) a method of quantifying the protein according to the        above (1) or the partial peptide according to the above (3), (6)        or (7), which comprises using the antibody according to (17);    -   (27) a pharmaceutical composition comprising the protein or salt        thereof according to the above (1) or the partial peptide or        salt thereof according to the above (3), (6) or (7);    -   (28) the pharmaceutical composition according to the above (27),        which is an agent for inhibiting tumor metastasis.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the comparison among human, mouse type 1, mouse type 2, andrat KiSS-1 proteins in terms of amino acid sequence. The identical aminoacids among these 4 different homologs are designated by asterisk. Aminoacids are represented by one-letter abbreviation.

BEST MODE FOR CARRYING OUT THE INVENTION

The protein of the present invention includes a human ligand proteinspecific to the above-mentioned novel human G protein-coupled receptorprotein (hOT7T175), rat and mouse homologs thereof, and mature formsthereof.

These proteins may be derived from any cells (e.g. splenocytes, nervecells, glial cells, β cells of pancreas, bone marrow cells, mesangialcells, Langerhans' cells, epidermic cells, epithelial cells, endothelialcells, fibroblasts, fibrocytes, myocytes, fat cells, immune cells (e.g.macrophage, T cells, B cells, natural killer cells, mast cells,neutrophil, basophil, eosinophil, monocyte), megakaryocyte, synovialcells, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary glandcells, hepatocytes or interstitial cells, the corresponding precursorcells, stem cells, cancer cells, etc.), blood cells, or any tissueswhere such cells are present, e.g. brain or any region of the brain(e.g. olfactory bulb, amygdaloid nucleus, basal ganglia, hippocampus,thalamus, hypothalamus, subthalamic nucleus, cerebral cortex, medullaoblongata, cerebellum, occipital pole, frontal lobe, temporal lobe,putamen, caudate nucleus, corpus callosum, substantia nigra), spinalcord, hypophysis, stomach, pancreas, kidney, liver, gonad, thyroid,gall-bladder, bone marrow, adrenal gland, skin, muscle, lung,gastrointestinal tract (e.g. large intestine and small intestine), bloodvessel, heart, thymus, spleen, submandibular gland, peripheral blood,peripheral blood cells, prostate, testis, ovary, placenta, uterus, bone,joint, skeletal muscle, etc. (especially brain or any of brain regions)of human and other mammals (e.g. guinea pigs, rats, mice, rabbits,swine, sheep, bovine, monkeys, etc.). The proteins may be a syntheticprotein.

The protein of the present invention has activity of binding toG-protein coupled receptor proteins in a specific manner. The G-proteincoupled receptor proteins include OT7T175 derived from a human, OT7T175derived from a rat, and their homologues derived from other mammals,such as a monkey and mouse. Examples of the protein of the presentinvention includes the type 1 and type 2 proteins derived from a mouse(comprising the amino acid sequence represented by SEQ ID NO:1 and NO:2,respectively), and the rat protein (comprising the amino acid sequencerepresented by SEQ ID NO:3), and also mature polypeptides of theseproteins. The mouse type 1 mature polypeptide refers to a protein havingthe amino acid sequence from 90th to 141th residues from the N-terminalof the amino acid sequence represented by SEQ ID NO:1. The mouse type 2mature polypeptide refers to a protein having the amino acid sequencefrom 94th to 145th residues from the N-terminal of the amino acidsequence represented by SEQ ID NO:2. The rat mature polypeptide refersto a protein having the amino acid sequence from 68th to 119th residuesfrom the N-terminal of the amino acid sequence represented by SEQ IDNO:3.

In this specification, “substantially the same amino acid sequence”refers to a variant of a reference amino acid sequence wherein (i) atleast one or more (preferably approximately 1 to 30, more preferablyapproximately 1 to 10, most preferably several (1 or 2)) amino acids aredeleted from the reference sequence; (ii) at least one or more(preferably approximately 1 to 30, more preferably approximately 1 to10, most preferably several (1 or 2)) amino acids are added to thereference sequence; (iii) at least one or more (preferably approximately1 to 30, more preferably approximately 1 to 10, most preferably several(1 or 2)) amino acids of the reference sequence are substituted withother amino acids; or (iv) any combination of these modifications isincluded in the reference sequence.

Specifically, for example, substantially the same amino acid sequence asthat represented by SEQ ID NO:1 or NO:3 includes an amino acid sequencehaving at least about 70% homology, preferably at least about 80%homology, more preferably at least about 90% homology, and mostpreferably at least about 95% homology to the amino acid sequencerepresented by SEQ ID NO:1 or NO:3.

Proteins which comprise substantially the same amino acid sequence asthat of the protein of the present invention, and ones which havesubstantially the same amino acid sequence as that of the protein of thepresent invention have substantially the same property as that of theprotein of the present invention. That is, these proteins have theproperty of binding to the G protein-coupled receptor proteins in aspecific manner. The receptors preferably include OT7T175 derived from ahuman (SEQ ID NO:7), OT7T175 derived from a rat (SEQ ID NO:8), andOT7T175 derived from a mouse (SEQ ID NO:24). In more details, saidproperty includes the binding activity to the G-protein coupled receptorproteins, the signal transduction activity, and the like. It ispreferred that the properties or activities of the above-mentionedproteins are equivalent (e.g., about 0.01- to 100-fold, preferably about0.5- to 20-fold, more preferably about 0.5- to 2-fold), but somequantitative factors, such as the level of activity, the molecularweight of the protein, may be different. These activities can bemeasured according to a publicly known method, for example, by thescreening method as described below.

In the present specification, the proteins of the present invention arerepresented in accordance with the conventional way of describingpeptides so as to place the N-terminus (amino terminus) on the left sideand the C-terminus (carboxyl terminus) on the right side. In the ligandproteins of the present invention, including the ligand proteincomprising the amino acid sequence shown by SEQ ID NO:1, the C-terminusis usually in the form of a carboxyl group (—COOH) or a carboxylate(—COO⁻) but may be in the form of an amide (—CONH₂) or an ester (—COOR).

Examples of the ester group shown by R include a C₁₋₆ alkyl group suchas methyl, ethyl, n-propyl, isopropyl, n-butyl, etc.; a C₃₋₈ cycloalkylgroup such as cyclopentyl, cyclohexyl, etc.; a C₆₋₁₂ aryl group such asphenyl, α-naphthyl, etc.; a C₇₋₁₄ aralkyl group such as aphenyl-C₁₋₂-alkyl group, e.g., benzyl, phenethyl, etc., or anα-naphthyl-C₁₋₂-alkyl group such as α-naphthylmethyl, etc.; and thelike. In addition, pivaloyloxymethyl or the like, which is used widelyas an ester for oral administration, may also be used.

When the ligand protein of the present invention has a carboxyl group(or a carboxylate) at a position other than the C-terminus, it may beamidated or esterified and such an amide or ester is also includedwithin the ligand protein of the present invention. The ester group maybe the same as described with respect to the C-terminus in the above.

Furthermore, the ligand proteins of the present invention includevariants of the proteins as described above wherein the amino group atthe N-terminal methionine residue is protected with a protecting group(for example, a C₁₋₆ acyl group such as a C₂₋₆ alkanoyl group, e.g.,formyl group, acetyl group, etc.); those wherein the N-terminal regionis cleaved in vivo to generate glutamyl group, which is thenpyroglutaminated; those wherein a substituent (e.g., —OH, —SH, aminogroup, imidazole group, indole group, guanidino group, etc.) on the sidechain of an amino acid in the molecule is protected with a suitableprotecting group (e.g., a C₁₋₆ acyl group such as a C₂₋₆ alkanoyl group,e.g., formyl group, acetyl group, etc.), or conjugated proteins such asglycoproteins which has sugar chains.

Examples of the ligand protein of the present invention includes theligand proteins derived from a mouse, comprising the amino acid sequencerepresented by SEQ ID NO:1 and NO:2 (mouse type 1 and type 2 ligands,respectively), and the ligand protein derived from a rat, comprising theamino acid sequence represented by SEQ ID NO:3 (rat ligand).

The present invention provides partial peptides of the ligand protein ofthe present invention (hereinafter sometimes referred to as the partialpeptides). These partial peptides may be any one derived from the ligandproteins of the present invention, but should have the property asdescribed above.

Specifically, partial peptides of the protein having the amino acidsequence represented by SEQ ID NO:1, NO:2 or NO:3 includes the matureforms as described above. The partial peptides of these mature partialpeptides are also included in the present invention as long as theyretain the property as described above.

Examples of partial peptides of the protein having the amino acidsequence represented by SEQ ID NO:1, NO:2 or NO:3 include:

-   -   (a) a polypeptide having the amino acid sequence from 90th to        141th residues from the N-terminal of the amino acid sequence        represented by SEQ ID NO:1, an amide or ester thereof, or a salt        thereof;    -   (b) a polypeptide comprising the amino acid sequence from 134th        to 141th residues from the N-terminal of the amino acid sequence        represented by SEQ ID NO:1 and consisting of 8 to 52 amino        acids, an amide or ester thereof, or a salt thereof;    -   (c) a polypeptide having the amino acid sequence from 94th to        145th residues from the N-terminal of the amino acid sequence        represented by SEQ ID NO:2, an amide or ester thereof, or a salt        thereof;    -   (d) a polypeptide comprising the amino acid sequence from 138th        to 145th residues from the N-terminal of the amino acid sequence        represented by SEQ ID NO:2 and consisting of 8 to 52 amino        acids, an amide or ester thereof, or a salt thereof;    -   (e) a polypeptide having the amino acid sequence from 68th to        119th residues from the N-terminal of the amino acid sequence        represented by SEQ ID NO:3, an amide or ester thereof, or a salt        thereof;    -   (f) a polypeptide comprising the amino acid sequence from 112th        to 119th residues from the N-terminal of the amino acid sequence        represented by SEQ ID NO:3 and consisting of 8 to 52 amino        acids, an amide or ester thereof, or a salt thereof;    -   (g) a polypeptide comprising the amino acid sequence from 132th        to 141th residues from the N-terminal of the amino acid sequence        represented by SEQ ID NO:1, an amide or ester thereof, or a salt        thereof;    -   (h) a polypeptide comprising the same or substantially the same        amino acid sequence as the sequence from 127th to 141th residues        from the N-terminal of the amino acid sequence represented by        SEQ ID NO:1, an amide or ester thereof, or a salt thereof;    -   (i) a polypeptide comprising the amino acid sequence from 110th        to 119th residues from the N-terminal of the amino acid sequence        represented by SEQ ID NO:3, an amide or ester thereof, or a salt        thereof;    -   (j) a polypeptide comprising the same or substantially the same        amino acid sequence as the sequence from 105th to 119th residues        from the N-terminal of the amino acid sequence represented by        SEQ ID NO:3, an amide or ester thereof, or a salt thereof.

Among the polypeptides as described in (a) to (j) above, those whereinthe C-terminal is an amide form are preferred.

“Substantially the same amino acid sequence” refers to amino acidsequences which have at least about 80% homology, preferably at leastabout 90% homology, more preferably at least about 95% homology, andmost preferably at least about 99% homology to the reference amino acidsequence.

Among those, preferred are the polypeptides, amides or esters thereof,or salts thereof described in (a) to (j) above, and more preferred are:

-   -   (i) a polypeptide having the amino acid sequence from 132th to        141th residues from the N-terminal of the amino acid sequence        represented by SEQ ID NO:1, an amide or ester thereof, or a salt        thereof;    -   (ii) a polypeptide having the amino acid sequence from 127th to        141th residues from the N-terminal of the amino acid sequence        represented by SEQ ID NO:1, an amide or ester thereof, or a salt        thereof;    -   (iii) a polypeptide having the amino acid sequence from 110th to        119th residues from the N-terminal of the amino acid sequence        represented by SEQ ID NO:3, an amide or ester thereof, or a salt        thereof;    -   (iv) a polypeptide having the amino acid sequence from 105th to        119th residues from the N-terminal of the amino acid sequence        represented by SEQ ID NO:3, an amide or ester thereof, or a salt        thereof.

Among these polypeptides, those wherein the C-terminal is an amide formare preferred.

The partial peptide of the present invention may have the amino acidsequences described above, from which at least 1 or 2 (preferablyapproximately 1 to 10, more preferably several (1 or 2)) amino acids aredeleted; to which at least 1 or 2 (preferably approximately 1 to 20,more preferably approximately 1 to 10, and even more preferably several(1 or 2)) amino acids are added; or, in which at least 1 or 2(preferably approximately 1 to 10, more preferably several (1 or 2))amino acids are substituted by other amino acids.

In the partial peptide of the present invention, the C-terminus isnormally a carboxyl group (—COOH) or carboxylate (—COO⁻) but theC-terminus may be in the form of an amide (—CONH₂) or an ester (—COOR),as described with regard to the ligand protein of the present invention(R has the same definition as above).

As described in the ligand protein of the present invention, the partialpeptide of the present invention further-includes those in which theamino group of the N-terminal methionine residue is protected by aprotecting group, those in which the N-terminal residue is cleaved invivo to generate glutamine residue, which is then pyroglutaminated,those in which substituents on the side chains of amino acids in themolecule are protected by appropriate protecting groups, and alsoconjugated peptides, such as so-called glycoproteins to which sugarchains are bound, and the like.

The salts of the ligand protein or the partial peptide of the presentinvention are formed with physiologically acceptable bases or acids.Especially, physiologically acceptable acid addition salts arepreferred. Examples of the salts include, for example, salts withinorganic acids (e.g., hydrochloric acid, phosphoric acid, hydrobromicacid, sulfuric acid); salts with organic acids (e.g., acetic acid,formic acid, propionic acid, fumaric acid, maleic acid, succinic acid,tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid,methanesulfonic acid, benzenesulfonic acid) and the like.

The ligand protein of the present invention or salts thereof may beproduced by a publicly known method for purifying a ligand protein fromcells or tissues of a human or other mammals as described above, or byculturing a transformant having the DNA encoding the ligand protein ofthe present invention, as described below. Furthermore, they may also beproduced by a method for synthesizing a protein, as described below, ormodified methods thereof.

When the ligand protein or salts thereof are produced from tissues orcells of a human or mammals, these tissues or cells are homogenized,then extracted with an acid or the like, and the extract is subjected toa combination of chromatography techniques, such as reverse phasechromatography, ion exchange chromatography, and the like to isolate andpurify the protein.

To synthesize the ligand protein of the present invention, the partialpeptide thereof, or salts or amides thereof, commercially availableresins for protein synthesis may be used. Examples of such resinsinclude chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin,aminomethyl resin, 4-benzyloxybenzyl alcohol resin,4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmehtylphenylacetamidomethyl resin, polyacrylamide resin,4-(2′,4′-dimethoxyphenylhydroxymethyl)phenoxy resin,4-(2′,4′-dimethoxyphenyl-Fmoc-aminoethyl)phenoxy resin, etc. Using theseresins, amino acids in which α-amino groups and functional groups on theside chains are appropriately protected are condensed on the resinaccording to the sequence of the target protein by various condensationmethods publicly known in the art. At the end of the reaction, theprotein is cut out from the resin and at the same time, the protectinggroups are removed. Then, intramolecular disulfide bond-forming reactionis performed in a highly diluted solution to obtain the target proteinor its amide.

For condensation of the protected amino acids described above, a varietyof activating reagents for protein synthesis may be used, andcarbodiimides are particularly preferable. Examples of suchcarbodiimides include DCC, N,N′-diisopropylcarbodiimide,N-ethyl-N′-(3-dimethylaminoprolyl)carbodiimide, etc. For activation bythese reagents, the protected amino acids in combination with aracemization inhibitor (e.g., HOBt, HOOBt) are added directly to theresin. Alternatively, the protected amino acids are previously activatedin the form of symmetric acid anhydrides, HOBt esters or HOOBt esters,and then thus activated protected amino acids are added to the resin.

Solvents suitable for use to activate the protected amino acids orcondense with the resin may be chosen from solvents known to be usablefor protein condensation reactions. Examples of such solvents are acidamides such as N,N-dimethylformamide, N,N-dimethylacetamide,N-methylpyrrolidone, etc.; halogenated hydrocarbons such as methylenechloride, chloroform, etc.; alcohols such as trifluoroethanol, etc.;sulfoxides such as dimethylsulfoxide, etc.; ethers such as pyridine,dioxane, tetrahydrofuran, etc.; nitrites such as acetonitrile,propionitrile, etc.; esters such as methyl acetate, ethyl acetate, etc.;and appropriate mixtures of these solvents. The reaction temperature isappropriately chosen from the range known to be applicable to proteinbinding reactions and is usually selected in the range of approximately−20° C. to 50° C. The activated amino acid derivatives are usedgenerally in an excess of 1.5 to 4 times. The condensation is examinedby a test using the ninhydrin reaction; when the condensation isinsufficient, the condensation can be completed by repeating thecondensation reaction without removal of the protecting groups. When thecondensation is yet insufficient even after repeating the reaction,unreacted amino acids are acetylated with acetic anhydride oracetylimidazole to avoid an adverse effect on the later reaction.

Examples of the protecting groups used to protect the amino groups ofthe starting compounds include Z, Boc, t-pentyloxycarbonyl,isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z,adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl,2-nitrophenylsulphenyl, diphenylphosphinothioyl, Fmoc, etc.

A carboxyl group can be protected by, e.g., alkyl esterification (in theform of linear, branched or cyclic alkyl esters of the alkyl moiety suchas methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, 2-adamantyl, etc.), aralkyl esterification(e.g., esterification in the form of benzyl ester, 4-nitrobenzyl ester,4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl ester, etc.),phenacyl esterification, benzyloxycarbonyl hydrazidation,t-butoxycarbonyl hydrazidation, trityl hydrazidation, or the like.

The hydroxyl group of serine can be protected through, for example, itsesterification or etherification. Examples of groups appropriately usedfor the esterification include a lower alkanoyl group, such as acetylgroup, an aroyl group such as benzoyl group, and a group derived fromcarbonic acid such as benzyloxycarbonyl group, ethoxycarbonyl group,etc. Examples of a group appropriately used for the etherificationinclude benzyl group, tetrahydropyranyl group, t-butyl group, etc.

Examples of groups for protecting the phenolic hydroxyl group oftyrosine include Bzl, C₁₂-Bzl, 2-nitrobenzyl, Br-Z, t-butyl, etc.

Examples of groups used to protect the imidazole moiety of histidineinclude Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP,benzyloxymethyl, Bum, Boc, Trt, Fmoc, etc.

Examples of the activated carboxyl groups in the starting compoundsinclude the corresponding acid anhydrides, azides, activated esters(esters with alcohols (e.g., pentachlorophenol, 2,4,5-trichlorophenol,2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB,N-hydroxysuccimide, N-hydroxyphthalimide, HOBt)). As the activated aminoacids, in which the amino groups are activated in the starting material,the corresponding phosphoric amides are employed.

To remove (eliminate) the protecting groups, there are used catalyticreduction under hydrogen gas flow in the presence of a catalyst such asPd-black or Pd-carbon; an acid treatment with anhydrous hydrogenfluoride, methanesulfonic acid, trifluoromethane-sulfonic acid ortrifluoroacetic acid, or a mixture solution of these acids; a treatmentwith a base such as diisopropylethylamine, triethylamine, piperidine orpiperazine; and reduction with sodium in liquid ammonia. The eliminationof the protecting group by the acid treatment described above is carriedout generally at a temperature of approximately −20° C. to 40° C. In theacid treatment, it is efficient to add a cation scavenger such asanisole, phenol, thioanisole, m-cresol, p-cresol, dimethylsulfide,1,4-butanedithiol or 1,2-ethanedithiol. Furthermore, 2,4-dinitrophenylgroup known as the protecting group for the imidazole of histidine isremoved by a treatment with thiophenol. Formyl group used as theprotecting group of the indole of tryptophan is eliminated by theaforesaid acid treatment in the presence of 1,2-ethanedithiol or1,4-butanedithiol, as well as by a treatment with an alkali such as adilute sodium hydroxide solution and dilute ammonia.

Protection of functional groups that should not be involved in thereaction of the starting materials, protecting groups, elimination ofthe protecting groups and activation of functional groups involved inthe reaction may be appropriately selected from publicly known groupsand publicly known means.

In another method for obtaining an amide of the protein, for example,the α-carboxyl group of the carboxy terminal amino acid is firstprotected by amidation; the peptide (protein) chain is then extendedfrom the amino group side to a desired length. Thereafter, a protein inwhich only the protecting group of the N-terminal α-amino group in thepeptide chain has been eliminated from the protein and a protein inwhich only the protecting group of the C-terminal carboxyl group hasbeen eliminated are prepared. The two proteins are condensed in amixture of the solvents described above. The details of the condensationreaction are the same as described above. After the protected proteinobtained by the condensation is purified, all the protecting groups areeliminated by the method described above to give the desired crudeprotein. This crude protein is purified by various known purificationmeans. Lyophilization of the major fraction gives the amide of thedesired protein.

To prepare the esterified protein, for example, the α-carboxyl group ofthe carboxy terminal amino acid is condensed with a desired alcohol toprepare the amino acid ester, which is followed by procedure similar tothe preparation of the amidated protein above to give the ester form ofthe desired protein.

The partial peptide of the ligand protein of the present invention canbe produced by publicly known methods for peptide synthesis, or bycleaving the protein of the present invention with an appropriatepeptidase. For the methods for peptide synthesis, for example, eithersolid phase synthesis or liquid phase synthesis may be used. That is,the partial peptide or amino acids that can construct the protein of thepresent invention are condensed with the remaining part. When theproduct contains protecting groups, these protecting groups are removedto give the desired peptide. Publicly known methods for condensation andelimination of the protecting groups are described in 1)-5) below.

-   -   1) M. Bodanszky & M. A. Ondetti: Peptide Synthesis, Interscience        Publishers, New York (1966)    -   2) Schroeder & Luebke: The Peptide, Academic Press, New York        (1965)    -   3) Nobuo Izumiya, et al.: Peptide Gosei-no-Kiso to Jikken        (Basics and experiments of peptide synthesis), published by        Maruzen Co. (1975)    -   4) Haruaki Yajima & Shunpei Sakakibara: Seikagaku Jikken Koza        (Biochemical Experiment) 1, Tanpakushitsu no Kagaku (Chemistry        of Proteins) IV, 205 (1977)    -   5) Haruaki Yajima, ed.: Zoku Iyakuhin no Kaihatsu (A sequel to        Development of Pharmaceuticals), Vol. 14, Peptide Synthesis,        published by Hirokawa Shoten

After completion of the reaction, the product may be purified andisolated by a combination of conventional purification methods such assolvent extraction, distillation, column chromatography, liquidchromatography and recrystallization to give the partial peptide of thepresent invention. When the partial peptide obtained by the abovemethods is in a free form, the peptide can be converted into anappropriate salt by a publicly known method; when the protein isobtained in a salt form, it can be converted into a free form by apublicly known method.

The polynucleotide encoding the ligand protein of the present inventionmay be any polynucleotide so long as it contains the base sequence (DNAor RNA, preferably DNA) encoding the ligand protein of the presentinvention described above. Such a polynucleotide may also be any one ofDNA or RNA such as mRNA encoding the ligand protein of the presentinvention, and may be double-stranded or single-stranded. When thepolynucleotide is double-stranded, it may be double-stranded DNA,double-stranded RNA or DNA:RNA hybrid. When the polynucleotide issingle-stranded, it may be a sense strand (i.e. a coding strand) or anantisense strand (i.e. a non-coding strand).

Using the polynucleotide encoding the ligand protein of the presentinvention, mRNA of the ligand protein of the present invention can bequantified by, for example, the publicly known method published in aseparate volume of Jikken Igaku 15(7) “New PCR and its application”(1997), or by its modifications.

The DNA encoding the ligand protein of the present invention may bederived from any of genomic DNA, genomic DNA library, cDNA derived fromthe cells and tissues described above, cDNA library derived from thecells and tissues described above and synthetic DNA. The vector to beused for the library may be any of bacteriophage, plasmid, cosmid andphagemid. The DNA may also be directly amplified by reversetranscriptase polymerase chain reaction (hereinafter abbreviated asRT-PCR) using the total RNA or mRNA fraction prepared from the cells andtissues described above.

Specifically, the DNA encoding the ligand protein of the presentinvention may be any DNA having the base sequence shown by SEQ ID NO:4,NO:5 or NO:6, or the base sequence hybridizable to the base sequencerepresented by SEQ ID NO:4, NO:5 or NO:6 under a high stringentcondition, and encoding a ligand protein having the activitiessubstantially equivalent to those of the ligand protein of the presentinvention (e.g., a ligand binding activity, a signal transductionactivity, etc.).

Specific examples of the DNA hybridizable to the base sequencerepresented by SEQ ID NO:4, NO:5 or NO:6 include DNA containing a basesequence having at least about 70% homology, preferably at least about80% homology, more preferably at least about 90% homology and mostpreferably at least about 95% homology to the base sequence representedby SEQ ID NO:4, NO:5 or NO:6.

The hybridization can be carried out by publicly known methods or bymodifications of these methods, for example, according to the methoddescribed in Molecular Cloning, 2nd (J. Sambrook et al., Cold SpringHarbor Lab. Press, 1989). A commercially available library may also beused according to the instructions of the attached manufacturer'sprotocol. Preferably, the hybridization can be carried out under a highstringent condition.

The high stringent condition refers to, for example, a sodiumconcentration of about 19 mM to about 40 mM, preferably about 19 mM toabout 20 mM and a temperature of about 50° C. to about 70° C.,preferably about 60° C. to about 65° C. In particular, the mostpreferred condition is a sodium concentration of about 19 mM and atemperature of about 65° C.

More specifically, the DNA encoding the ligand protein having the aminoacid sequence represented by SEQ ID NO:1 includes DNA having the basesequence represented by SEQ ID NO:4. The DNA encoding the ligand proteinhaving the amino acid sequence represented by SEQ ID NO:2 includes DNAhaving the base sequence represented by SEQ ID NO:5. The DNA encodingthe ligand protein having the amino acid sequence represented by SEQ IDNO:3 includes DNA having the base sequence represented by SEQ ID NO:6.

The polynucleotide comprising a part of the base sequence of the DNAencoding the ligand protein of the present invention or a part of thebase sequence complementary to the DNA is intended to include not onlythe DNA encoding the partial peptide of the present invention describedbelow, but also RNA.

According to the present invention, antisense polynucleotides (nucleicacids) that can inhibit the replication or expression of genes of theligand proteins can be designed and synthesized based on the basesequence information of the cloned or isolated DNAs encoding theproteins. Such a polynucleotide (nucleic acid) is capable of hybridizingto RNA of the gene of the ligand protein to inhibit the synthesis orfunction of said RNA, or capable of interact with RNA associated withthe ligand protein to modulate or control the expression of the gene ofthe ligand protein. Polynucleotides which is complementary to theselected sequence region of RNA associated with the ligand protein andones which is specifically hybridizable to RNA associated with theligand protein are useful in modulating or controlling the expression ofgene of the ligand protein in vivo and in vitro, and thus useful for thetreatment or diagnosis of diseases. The term “corresponding” means to behomologous or complementary to a particular nucleotide sequence, basesequence or nucleic acid sequence including a gene. The “corresponding”relation between a nucleotide sequence, base sequence or nucleic acidsequence and a peptide (protein) usually means that amino acids of thepeptide (protein) is under control of the nucleotide (nucleic acid)sequence or their complementary sequence. In the gene of ligand protein,the 5′-end hairpin loop, 5′-end 6-base-pair repeats, 5′-end untranslatedregion, polypeptide translation initiation codon, protein coding region,ORF translation initiation codon, 3′-end untranslated region, 3′-endpalindrome region, and 3′-end hairpin loop, may be selected as preferredtarget regions, though any other region may be selected as a target inthe gene of ligand protein.

The relationship between the targeted nucleic acids and thepolynucleotides complementary to at least a part of the target,specifically the relationship between the target and the polynucleotideshybridizable to the target, can be denoted to be “antisense”. Examplesof the antisense polynucleotides include polydeoxynucleotides containing2-deoxy-D-ribose, polydeoxynucleotides containing D-ribose, any othertype of polynucleotides which are N-glycosides of a purine or pyrimidinebase, or other polymers containing non-nucleotide backbones (e.g.,protein nucleic acids and synthetic sequence-specific nucleic acidpolymers commercially available) or other polymers containingnonstandard linkages (provided that the polymers contain nucleotideshaving such a configuration that allows base pairing or base stacking,as is found in DNA or RNA), etc. The antisense polynucleotides may bedouble-stranded DNA, single-stranded DNA, single-stranded RNA or aDNA:RNA hybrid, and may further include unmodified polynucleotides (orunmodified oligonucleotides), those with publicly known types ofmodifications, for example, those with labels known in the art, thosewith caps, methylated polynucleotides, those with substitution of one ormore naturally occurring nucleotides by their analogue, those withintramolecular modifications of nucleotides such as those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates,carbamates, etc.) and those with charged linkages or sulfur-containinglinkages (e.g., phosphorothioates, phosphorodithioates, etc.), thosehaving side chain groups such as proteins (nucleases, nucleaseinhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.),saccharides (e.g., monosaccharides, etc.), those with intercalators(e.g., acridine, psoralen, etc.), those containing chelators (e.g.,metals, radioactive metals, boron, oxidative metals, etc.), thosecontaining alkylating agents, those with modified linkages (e.g., aanomeric nucleic acids, etc.), and the like. Herein the terms“nucleoside”, “nucleotide” and “nucleic acid” are used to refer tomoieties that contain not only the purine and pyrimidine bases, but alsoother heterocyclic bases, which have been modified. Such modificationsmay include methylated purines and pyrimidines, acylated purines andpyrimidines and other heterocyclic rings. Modified nucleotides andmodified nucleotides also include modifications on the sugar moiety,wherein, for example, one or more hydroxyl groups may optionally besubstituted with a halogen atom(s), an aliphatic group(s), etc., or maybe converted into the corresponding functional groups such as ethers,amines, or the like.

The antisense polynucleotide (nucleic acid) of the present invention isRNA, DNA or a modified nucleic acid (RNA, DNA). Specific examples of themodified nucleic acid are, but not limited to, sulfur and thiophosphatederivatives of nucleic acids and those resistant to degradation ofpolynucleoside amides or oligonucleoside amides. The antisense nucleicacids of the present invention can be modified preferably based on thefollowing design, that is, by increasing the intracellular stability ofthe antisense nucleic acid, increasing the cellular permeability of theantisense nucleic acid, increasing the affinity of the nucleic acid tothe targeted sense strand to a higher level, or minimizing the toxicity,if any, of the antisense nucleic acid.

Many of such modifications are known in the art, as disclosed in J.Kawakami, et al., Pharm. Tech. Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp.395, 1992; S. T. Crooke, et al. ed., Antisense Research andApplications, CRC Press, 1993; etc.

The antisense nucleic acid of the present invention may contain alteredor modified sugars, bases or linkages. The antisense nucleic acid mayalso be provided in a specialized form such as liposomes, microspheres,or may be applied to gene therapy, or may be provided in combinationwith attached moieties. Such attached moieties-include polycations suchas polylysine that act as charge neutralizers of the phosphate backbone,or hydrophobic moieties such as lipids (e.g., phospholipids,cholesterols, etc.) that enhance the interaction with cell membranes orincrease uptake of the nucleic acid. Preferred examples of the lipids tobe attached are cholesterols or derivatives thereof (e.g., cholesterylchloroformate, cholic acid, etc.). These moieties may be attached to thenucleic acid at the 3′ or 5′ ends thereof and may also be attachedthereto through a base, sugar, or intramolecular nucleoside linkage.Other moieties may be capping groups specifically placed at the 3′ or 5′ends of the nucleic acid to prevent degradation by nucleases such asexonuclease, RNase, etc. Such capping groups include, but are notlimited to, hydroxyl protecting groups known in the art, includingglycols such as polyethylene glycol, tetraethylene glycol and the like.

The inhibitory activity of the antisense nucleic acid can be examinedusing the transformant of the present invention, the gene expressionsystem of the present invention in vivo and in vitro, or the translationsystem of the ligand protein in vivo and in vitro. The nucleic acid canbe applied to cells by a variety of publicly known methods.

The DNA encoding the partial peptide of the present invention may be anyDNA so long as it contains the base sequence encoding the partialpeptide of the present invention described above. The DNA may also bederived from any of genomic DNA, genomic DNA library, cDNA derived fromthe cells and tissues described above, cDNA library derived from thecells and tissues described above and synthetic DNA. The vector to beused for the library may be derived from any of bacteriophage, plasmid,cosmid and phagemid. The DNA may also be directly amplified by reversetranscriptase polymerase chain reaction (hereinafter abbreviated asRT-PCR) using mRNA fraction prepared from the cells and tissuesdescribed above.

Specifically, the DNA encoding the partial peptide of the presentinvention may be any one of, for example, (1) DNA containing a partialbase sequence of the DNA having the base sequence represented by SEQ IDNO:4, NO:5 or NO:6, or (2) any DNA containing a partial base sequence ofthe DNA having a base sequence hybridizable to the base sequencerepresented by SEQ ID NO:4, NO:5 or NO:6 under a high stringentcondition and encoding a ligand protein which has the activities (e.g.,a ligand-biding activity, a signal transduction activity, etc.)substantially equivalent to those of the ligand protein of the presentinvention. Typical examples of DNA containing a partial base sequence ofthe DNA having the base sequence represented by SEQ ID NO:4, NO:5 orNO:6 include DNAs encoding the mature forms of the ligand proteins ofthe present invention.

Examples of DNA that is hybridizable to the base sequence represented bySEQ ID NO:4, NO:5 or NO:6 include DNA containing a base sequence havingat least about 70% homology, preferably at least about 80% homology,more preferably at least about 90% homology and most preferably at leastabout 95% homology to the base sequence represented by SEQ ID NO:4, NO:5or NO:6.

For cloning of the DNA that completely encodes the ligand protein of thepresent invention or its partial peptide (hereinafter sometimescollectively referred to as the ligand protein of the presentinvention), the DNA may be either amplified by PCR using synthetic DNAprimers containing a part of the base sequence of the ligand protein ofthe present invention, or the DNA inserted into an appropriate vectorcan be selected by hybridization with a labeled DNA fragment orsynthetic DNA that encodes a part or entire region of the ligand proteinof the present invention. The hybridization can be carried out, forexample, according to the method described in Molecular Cloning, 2nd, J.Sambrook et al., Cold Spring Harbor Lab. Press, 1989. The hybridizationmay also be performed using commercially available library in accordancewith the protocol described in the attached instructions.

Conversion of the base sequence of the DNA can be effected by the PCRmethod or a publicly known method such as the Gapped duplex method orthe Kunkel method or its modification, using a publicly known kit suchas Mutan™-K super Express Km (Takara Shuzo Co., Ltd.) or Mutan™-K(Takara Shuzo Co., Ltd.).

The cloned DNA encoding the ligand protein can be used, depending uponpurpose, as it is or if desired, for example, after digestion with arestriction enzyme or after addition of a linker thereto. The DNA maycontain ATG as a translation initiation codon at the 5′ end thereof andmay further contain TAA, TGA or TAG as a translation termination codonat the 3′ end thereof. These translation initiation and terminationcodons may also be added by using an appropriate synthetic DNA adapter.

The expression vector for the ligand protein of the present inventioncan be produced, for example, by (a) excising the desired DNA fragmentfrom the DNA encoding the ligand protein of the present invention, andthen (b) ligating the DNA fragment with an appropriate expression vectordownstream a promoter in the vector.

Examples of the vector include plasmids derived form E. coli (e.g.,pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis(e.g., pUB110, pTP5, pC194), plasmids derived from yeast (e.g., pSH19,pSH15), bacteriophages such as λ-phage, etc., animal viruses such asretrovirus, vaccinia virus, baculovirus, etc. as well as pA1-11, pXT1,pRc/CMV, pRc/RSV, pcDNAI/Neo, etc.

The promoter used in the present invention may be any promoter if itmatches well with a host to be used for gene expression. In the case ofusing animal cells as the host, examples of the promoter include SRαpromoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter,etc. Among them, CMV promoter or SRa promoter is preferably used.

When the host is bacteria of the genus Escherichia, preferred examplesof the promoter include trp promoter, lac promoter, recA promoter,λP_(L) promoter, 1pp promoter, etc. In the case of using bacteria of thegenus Bacillus as the host, preferred example of the promoter are SPO1promoter, SPO2 promoter and penP promoter. When yeast is used as thehost, preferred examples of the promoter are PHO5 promoter, PGKpromoter, GAP promoter and ADH promoter. When insect cells are used asthe host, preferred examples of the promoter include polyhedrin prompterand P10 promoter.

In addition to the foregoing examples, the expression vector may furtheroptionally contain an enhancer, a splicing signal, a poly A additionsignal, a selection marker, SV40 replication origin (hereinaftersometimes abbreviated as SV400ri) etc. Examples of the selection markerinclude dihydrofolate reductase (hereinafter sometimes abbreviated asdhfr) gene [methotrexate (MTX) resistance], ampicillin resistant gene(hereinafter sometimes abbreviated as Amp^(r)), neomycin resistant gene(hereinafter sometimes abbreviated as Neo^(r), G418 resistance), etc. Inparticular, when dhfr gene is used as the selection marker in CHO(dhfr⁻) cells, selection can also be made on thymidine free media.

If necessary and desired, a signal sequence that matches with a host isadded to the N-terminus of the ligand protein of the present invention.Examples of the signal sequence that can be used are Pho A signalsequence, OmpA signal sequence, etc. in the case of using bacteria ofthe genus Escherichia as the host; α-amylase signal sequence, subtilisinsignal sequence, etc. in the case of using bacteria of the genusBacillus as the host; MFα signal sequence, SUC2 signal sequence, etc. inthe case of using yeast as the host; and insulin signal sequence,α-interferon signal sequence, antibody molecule signal sequence, etc. inthe case of using animal cells as the host, respectively.

Using the vector containing the DNA encoding the receptor protein of thepresent invention thus constructed, transformants can be produced.

Examples of the host, which may be employed, are bacteria belonging tothe genus Escherichia, bacteria belonging to the genus Bacillus, yeast,insect cells, insects and animal cells, etc.

Specific examples of the bacteria belonging to the genus Escherichiainclude Escherichia coli K12 DH1 (Proc. Natl. Acad. Sci. U.S.A., 60, 160(1968)), JM103 (Nucleic Acids Research, 9, 309 (1981)), JA221 (Journalof Molecular Biology, 120, 517 (1978)), HB101 (Journal of MolecularBiology, 41, 459 (1969)), C600 (Genetics, 39, 440 (1954)), etc.

Examples of the bacteria belonging to the genus Bacillus includeBacillus subtilis MI114 (Gene, 24, 255 (1983)), 207-21 (Journal ofBiochemistry, 95, 87 (1984)), etc.

Examples of yeast include Saccharomyces cereviseae AH22, AH22R⁻,NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036,Pichia pastoris KM71, etc.

Examples of insect cells include, for the virus AcNPV, Spodopterafrugiperda cells (Sf cells), MG1 cells derived from mid-intestine ofTrichoplusia ni, High Five™ cells derived from egg of Trichoplusia ni,cells derived from Mamestra brassicae, cells derived from Estigmenaacrea, etc.; and for the virus BmNPV, Bombyx mori N cells (BmN cells),etc. are used. Examples of the Sf cell which can be used are Sf9 cells(ATCC CRL1711) and Sf21 cells (both cells are described in Vaughn, J. L.et al., In Vivo, 13, 213-217 (1977).

As the insect, for example, a larva of Bombyx mori can be used (Maeda,et al., Nature, 315, 592 (1985)).

Examples of animal cells include monkey cells COS-7, Vero, Chinesehamster cells CHO (hereinafter referred to as CHO cells), dhfr genedeficient Chinese hamster cells CHO (hereinafter simply referred to asCHO(dhfr⁻) cell), mouse L cells, mouse AtT-20, mouse myeloma cells, ratGH3, human FL cells, etc.

Bacteria belonging to the genus Escherichia can be transformed, forexample, by the method described in Proc. Natl. Acad. Sci. U.S.A., 69,2110 (1972) or Gene, 17, 107 (1982).

Bacteria belonging to the genus Bacillus can be transformed, forexample, by the method described in Molecular & General Genetics, 168,111 (1979).

Yeast can be transformed, for example, by the method described inMethods in Enzymology, 194, 182-187 (1991), Proc. Natl. Acad. Sci.U.S.A., 75, 1929 (1978), etc.

Insect cells or insects can be transformed, for example, according tothe method described in Bio/Technology, 6, 47-55 (1988), etc.

Animal cells can be transformed, for example, according to the methoddescribed in Saibo Kogaku (Cell Engineering), extra issue 8, Shin SaiboKogaku Jikken Protocol (New Cell Engineering Experimental Protocol),263-267 (1995), published by Shujunsha, or Virology, 52, 456 (1973).

Thus, the transformant transformed with the expression vector containingthe DNA encoding the ligand protein of the present invention can beobtained.

When the host is bacteria belonging to the genus Escherichia or thegenus Bacillus, the transformant can be appropriately incubated in aliquid medium which contains materials required for growth of thetransformant such as carbon sources, nitrogen sources, inorganicmaterials, and so on. Examples of the carbon sources include glucose,dextrin, soluble starch, sucrose, etc. Examples of the nitrogen sourcesinclude inorganic or organic materials such as ammonium salts, nitratesalts, corn steep liquor, peptone, casein, meat extract, soybean cake,potato extract, etc. Examples of the inorganic materials are calciumchloride, sodium dihydrogenphosphate, magnesium chloride, etc. Inaddition, yeast extracts, vitamins, growth promoting factors etc. mayalso be added to the medium. Preferably, pH of the medium is adjusted toabout 5 to about 8.

A preferred example of the medium for incubation of the bacteriabelonging to the genus Escherichia is M9 medium supplemented withglucose and Casamino acids (Miller, Journal of Experiments in MolecularGenetics, 431-433, Cold Spring Harbor Laboratory, New York, 1972). Ifnecessary and desired, a chemical such as 3β-indolylacrylic acid can beadded to the medium thereby to activate the promoter efficiently.

When the bacteria belonging to the genus Escherichia are used as thehost, the transformant is usually cultivated at about 15° C. to about43° C. for about 3 hours to about 24 hours. If necessary and desired,the culture may be aerated or agitated.

When the bacteria belonging to the genus Bacillus are used as the host,the transformant is cultivated generally at about 30° C. to about 40° C.for about 6 hours to about 24 hours. If necessary and desired, theculture can be aerated or agitated.

When yeast is used as the host, the transformant is cultivated, forexample, in Burkholder's minimal medium (Bostian, K. L. et al., Proc.Natl. Acad. Sci. U.S.A., 77, 4505 (1980)) or in SD medium supplementedwith 0.5% Casamino acids (Bitter, G. A. et al., Proc. Natl. Acad. Sci.U.S.A., 81, 5330 (1984)). Preferably, pH of the medium is adjusted toabout 5 to about 8. In general, the transformant is cultivated at about20° C. to about 35° C. for about 24 hours to about 72 hours. Ifnecessary and desired, the culture can be aerated or agitated.

When insect cells or insects are used as the host, the transformant iscultivated in, for example, Grace's Insect Medium (Grace, T. C. C.,Nature, 195, 788 (1962)) to which an appropriate additive such asimmobilized 10% bovine serum is added. Preferably, pH of the medium isadjusted to about 6.2 to about 6.4. Normally, the transformant iscultivated at about 27° C. for about 3 days to about 5 days and, ifnecessary and desired, the culture can be aerated or agitated.

When animal cells are employed as the host, the transformant iscultivated in, for example, MEM medium containing about 5% to about 20%fetal bovine serum (Science, 122, 501 (1952)), DMEM medium (Virology, 8,396 (1959)), RPMI 1640 medium (The Journal of the American MedicalAssociation, 199, 519 (1967)), 199 medium (Proceeding of the Society forthe Biological Medicine, 73, 1 (1950)), etc. Preferably, pH of themedium is adjusted to about 6 to about 8. The transformant is usuallycultivated at about 30° C. to about 40° C. for about 15 hours to about60 hours and, if necessary and desired, the culture can be aerated oragitated.

As described above, the ligand protein of the present invention or thepartial peptide thereof can be produced in the cell membrane of thetransformant.

The ligand protein of the present invention or the partial peptidethereof can be separated and purified from the culture described aboveby the following procedures.

When the ligand protein of the present invention or the partial peptidethereof is extracted from the culture or cells, after cultivation, thetransformants or cells are collected by a publicly known method andsuspended in a appropriate buffer. The transformants or cells are thendisrupted by publicly known methods such as ultrasonication, a treatmentwith lysozyme and/or freeze-thaw cycling, followed by centrifugation,filtration, etc. Thus, the crude extract of the ligand protein of thepresent invention or the partial peptide thereof can be obtained. Thebuffer used for the procedures may contain a protein modifier such asurea or guanidine hydrochloride, or a surfactant such as Triton X-100™,etc. When the ligand protein or the partial peptide thereof is secretedin the culture, after completion of the cultivation, the supernatant canbe separated from the transformants or cells, and then can be collectedby a publicly known method.

The ligand protein of the present invention or the partial peptidethereof contained in the supernatant or the extract thus obtained can bepurified by appropriately combining publicly known methods forseparation and purification. Such publicly known methods for separationand purification include a method utilizing difference in solubilitysuch as salting out, solvent precipitation, etc.; a method utilizingmainly difference in molecular weight such as dialysis, ultrafiltration,gel filtration, SDS-polyacrylamide gel electrophoresis, etc.; a methodutilizing difference in electric charge such as ion exchangechromatography, etc.; a method utilizing difference in specific affinitysuch as affinity chromatography, etc.; a method utilizing difference inhydrophobicity such as reverse phase high performance liquidchromatography, etc.; a method utilizing difference in isoelectric pointsuch as isoelectrofocusing electrophoresis; and the like.

When the thus obtained ligand protein or the partial peptide thereof isin a free form, it can be converted into the salt by publicly knownmethods or modifications thereof. On the other hand, when the receptorprotein is obtained in the form of a salt, it can be converted into thefree form or in the form of a different salt by publicly known methodsor modifications thereof.

The ligand protein or the partial peptide thereof produced by therecombinant can be treated, before or after the purification, with anappropriate protein modifying enzyme so that the protein can beappropriately modified or be deprived of its partial polypeptide.Examples of the protein-modifying enzyme include trypsin, chymotrypsin,arginyl endopeptidase, protein kinase, glycosidase or the like.

The activity of the thus produced ligand protein of the presentinvention or salts thereof, the partial peptide of the presentinvention, an ester or amide thereof, or a salt thereof can bedetermined by a binding assay to a labeled receptor, an enzymeimmunoassay using a specific antibody, or the like.

Antibodies to the ligand protein of the present invention or saltsthereof, the partial peptide of the present invention, an ester or amidethereof, or a salt thereof (hereinafter sometimes referred to as theligand protein of the present invention) may be any polyclonal ormonoclonal antibodies, as long as they are capable of recognizing theligand protein of the present invention.

The antibodies to the ligand protein of the present invention may beproduced by publicly known methods for producing antibodies or antisera,using as antigens the ligand protein of the present invention.

[Preparation of Monoclonal Antibody]

(a) Preparation of Monoclonal Antibody-Producing Cells

A receptor to the ligand protein of the present invention (hereinaftersometimes referred to as “the receptor”) may be administered to mammalseither alone or together with carriers or diluents at the site where theproduction of antibody is possible by the administration. In order topotentiate the antibody productivity upon the administration, completeFreund's adjuvants or incomplete Freund's adjuvants may be administered.The administration is usually carried out once in every two to six weeksand 2 to 10 times in total. Examples of the applicable mammals aremonkeys, rabbits, dogs, guinea pigs, mice, rats, sheep and goats, withmice and rats being preferred.

In the preparation of monoclonal antibody-producing cells, warm-bloodedanimals, e.g., mice, immunized with an antigen wherein the antibodytiter is noted is selected, then the spleen or lymph node is collectedafter 2 to 5 days from the final immunization and antibody-producingcells contained therein are fused with myeloma cells to give monoclonalantibody-producing hybridomas. Measurement of the antibody titer inantisera may be made, for example, by reacting a labeled ligand protein,as described below, with the antiserum, followed by assaying theactivity of the labeling agent bound to the antibody. The fusion may beoperated, for example, by the known Koehler and Milstein method (Nature,256, 495, 1975). Examples of the fusion accelerator are polyethyleneglycol (PEG), Sendai virus, etc., of which PEG is preferably employed.

Examples of the myeloma cells are NS-1, P3U1, SP2/0, etc. In particular,P3U1 is preferably employed. A preferred ratio of the count of theantibody-producing cells used (spleen cells) to the count of myelomacells is within a range of approximately 1:1 to 20:1. When PEG(preferably, PEG 1000 to PEG 6000) is added in a concentration ofapproximately 10 to 80% followed by incubating at about 20 to about 40°C., preferably at about 30 to about 37° C. for about 1 to about 10minutes, an efficient cell fusion can be carried out.

Various methods can be used for screening of a monoclonalantibody-producing hybridoma. Such methods include a method whichcomprises adding the supernatant of hybridoma to a solid phase (e.g.,microplate) adsorbed with the ligand protein of the present invention asan antigen directly or together with a carrier, adding ananti-immunoglobulin antibody (when mouse cells are used for the cellfusion, anti-mouse immunoglobulin antibody is used) labeled with aradioactive substance or an enzyme, or Protein A and detecting themonoclonal antibody bound to the solid phase, and a method whichcomprises adding the supernatant of hybridoma to a solid phase adsorbedwith an anti-immunoglobulin antibody or Protein A, adding the ligandprotein labeled with a radioactive substance or an enzyme and detectingthe monoclonal antibody bound to the solid phase.

The monoclonal antibody can be selected by publicly known methods or bymodifications of these methods. In general, the selection can beeffected in a medium for animal cells supplemented with HAT(hypoxanthine, aminopterin and thymidine). Any selection and growthmedium can be employed as far as the hybridoma can grow therein. Forexample, RPMI 1640 medium containing 1% to 20%, preferably 10% to 20%fetal bovine serum, GIT medium (Wako Pure Chemical Industries, Ltd.)containing 1% to 10% fetal bovine serum, a serum free medium forcultivation of a hybridoma (SFM-101, Nissui Seiyaku Co., Ltd.) and thelike can be used for the selection and growth medium. The cultivation iscarried out generally at 20° C. to 40° C., preferably at about 37° C.,for 5 days to 3 weeks, preferably 1 to 2 weeks. The cultivation can beconducted normally in 5% CO₂. The antibody titer of the culturesupernatant of hybridomas can be determined as in the assay for theantibody titer in antisera described above.

(b) Purification of Monoclonal Antibody

Separation and purification of a monoclonal antibody can be carried outby methods applied to conventional separation and purification ofimmunoglobulins, as in the conventional methods for separation andpurification of polyclonal antibodies (e.g., salting-out, alcoholprecipitation, isoelectric point precipitation, electrophoresis,adsorption and desorption with ion exchangers (e.g., DEAE),ultracentrifugation, gel filtration, or a specific purification methodwhich comprises collecting only an antibody with an activated adsorbentsuch as an antigen-binding solid phase, Protein A, Protein G, etc. anddissociating the binding to obtain the antibody).

[Preparation of Polyclonal Antibody]

The polyclonal antibody of the present invention can be produced bypublicly known methods or modifications thereof. For example, a complexof an immunogen (the ligand protein as an antigen) and a carrier proteinis prepared, and a mammal is immunized with the complex in a mannersimilar to the method described above for the production of monoclonalantibodies. Materials containing the antibody to the ligand protein orthe receptor protein of the present invention is collected from theimmunized animal, followed by separation and purification of theantibody.

In the complex of an immunogen and a carrier protein used to immunize amammal, the type of carrier protein and the mixing ratio of a carrier tohapten may be any type and in any ratio, as long as the antibody isefficiently produced to the hapten immunized by crosslinking to thecarrier. For example, bovine serum albumin, bovine thyroglobulins,keyhole limpet hemocyanin, etc. is coupled to hapten in acarrier-to-hapten weight ratio of approximately 0.1 to 20, preferablyabout 1 to about 5.

A variety of condensing agents can be used for the coupling of a carrierto hapten. Glutaraldehyde, carbodiimide, maleimide activated ester,activated ester reagents containing thiol group or dithiopyridyl group,etc. are used for the coupling.

The condensation product is administered to warm-blooded animals eithersolely or together with carriers or diluents to the site in which theantibody can be produce by the administration. In order to potentiatethe antibody productivity upon the administration, complete Freund'sadjuvant or incomplete Freund's adjuvant may be administered. Theadministration is usually made once approximately in every 2 to 6 weeksand about 3 to about 10 times in total.

The polyclonal antibody can be collected from the blood, ascites, etc.,preferably from the blood of mammals immunized by the method describedabove.

The titer of polyclonal antibody in antiserum can be assayed by the sameprocedure as that for the determination of antibody titer in serumdescribed above. The separation and purification of the polyclonalantibody can be carried out, following the method for the separation andpurification of immunoglobulins performed as applied to the separationand purification of monoclonal antibodies described hereinabove.

The ligand protein of the present invention or salts thereof, thepartial peptide of the present invention, an ester or amide thereof, ora salt thereof, and the DNA encoding it can be used for: <1> aprophylactic and/or therapeutic agent for diseases associated withdysfunction of the ligand protein of the present invention, <2> an agentfor genetic diagnosis, <3> quantification of a receptor to the ligandprotein of the present invention, <4> screening for a compound (anagonist, an antagonist, etc.) that alters the binding property betweenthe ligand protein of the present invention and its receptor, <5> aprophylactic and/or therapeutic agent for various diseases comprising acompound (an agonist or an antagonist) that alters the binding propertybetween the ligand protein of the present invention and its receptor,<6> quantification of the ligand protein of the present invention, <7>neutralization by an antibody to the ligand protein of the presentinvention, and <8> preparation of non-human animals that possess the DNAencoding the ligand protein of the present invention.

In particular, by use of the receptor binding assay, as described below,using the expression system of the recombinant ligand protein of thepresent invention, a compound (e.g., an agonist, an antagonist) thatalters the binding property of the human- or mammal-specific receptor tothe ligand protein of the present invention can be screened, and theagonist or antagonist can be used as a prophylactic and therapeuticagent for various diseases.

Use of the ligand protein of the present invention, the DNA encoding it(hereinafter sometimes referred to as the DNA of the present invention)and the antibody to the ligand protein of the present invention(hereinafter sometimes referred to as the antibody of the presentinvention) is specifically described in the following.

<1> A Prophylactic and/or Therapeutic Agent for Diseases Associated withDysfunction of the Ligand Protein of the Present Invention

The ligand protein of the present invention, or the DNA encoding theligand protein can be used as a prophylactic and/or therapeutic agentfor diseases associated with dysfunction of the ligand protein of thepresent invention.

For example, when a patient cannot rely on the physiological activity ofthe receptor due to a decrease in the ligand protein of the presentinvention (deficiency of the ligand protein), the amount of the ligandprotein in the patient can be increased and the activity of the receptorto the ligand can be sufficiently induced by (i) administering theligand protein of the present invention to the patient to supplement theamount of the ligand protein; or (ii) (a) administering the DNA encodingthe ligand protein to express the same in the patient; or (b)introducing and expressing the DNA encoding the ligand protein in targetcells, and then transplanting the cells to the patient. Thus, the ligandprotein of the present invention or the DNA encoding the ligand proteinis useful as a safe and low toxic prophylactic and/or therapeutic agentfor diseases associated with dysfunction of the ligand protein of thepresent invention.

The ligand protein of the present invention and the DNA encoding theligand protein have the activity of inhibiting tumor metastasis, andthus are useful for the prevention and/or treatment of any type ofcancers (e.g., cancers of lung, stomach, liver, pancreas, largeintestine, rectum, colon, prostate, ovary, uterine cervix, breast, etc.)

Furthermore, an agonist of the receptor protein has the activity ofregulating placental function, and thus is useful for the preventionand/or treatment of choriocarcinoma, hydatid mole, invasive mole,miscarriage, fetal dysgenesis, dysbolism of saccharide, dysbolism oflipid, or induction of delivery.

When the ligand protein of the present invention is used as theprophylactic/therapeutic agent, the ligand protein can be prepared intoa pharmaceutical composition in a conventional manner.

On the other hand, where the DNA encoding the ligand protein of thepresent invention (hereinafter sometimes referred to as the DNA of thepresent invention) is used as the prophylactic/therapeutic agentdescribed above, the DNA itself is administered; alternatively, the DNAis inserted into an appropriate vector such as retrovirus vector,adenovirus vector, adenovirus-associated virus vector, etc. and thenadministered in a conventional manner. The DNA of the present inventionmay also be administered as naked DNA, or with adjuvants to assist itsuptake by gene gun or through a catheter such as a catheter with ahydrogel.

For example, (i) the ligand protein of the present invention or (ii) theDNA encoding the ligand protein can be used orally, for example, in theform of tablets which may be sugar coated if necessary and desired,capsules, elixirs, microcapsules etc., or parenterally in the form ofinjectable preparations such as a sterile solution and a suspension inwater or with other pharmaceutically acceptable liquid. Thesepreparations can be produced by mixing (i) the ligand protein of thepresent invention or (ii) the DNA encoding the ligand protein with aphysiologically acceptable known carrier, a flavoring agent, anexcipient, a vehicle, an antiseptic agent, a stabilizer, a binder, etc.in a unit dosage form required in a generally accepted manner that isapplied to making pharmaceutical preparations. The effective componentin the preparation is controlled in such a dose that an appropriate doseis obtained within the specified range given.

Additives miscible with tablets, capsules, etc. include a binder such asgelatin, corn starch, tragacanth and gum arabic, an excipient such ascrystalline cellulose, a swelling agent such as corn starch, gelatin andalginic acid, a lubricant such as magnesium stearate, a sweetening agentsuch as sucrose, lactose and saccharin, and a flavoring agent such aspeppermint, akamono oil and cherry. When the unit dosage is in the formof capsules, liquid carriers such as oils and fats may further be usedtogether with the additives described above. A sterile composition forinjection may be formulated by conventional procedures used to makepharmaceutical compositions, e.g., by dissolving or suspending theactive ingredients in a vehicle such as water for injection with anaturally occurring vegetable oil such as sesame oil and coconut oil,etc. to prepare the pharmaceutical composition. Examples of an aqueousmedium for injection include physiological saline and an isotonicsolution containing glucose and other auxiliary agents (e.g.,D-sorbitol, D-mannitol, sodium chloride) and may be used in combinationwith an appropriate dissolution aid such as an alcohol (e.g., ethanol orthe like), a polyalcohol (e.g., propylene glycol and polyethyleneglycol), a nonionic surfactant (e.g., polysorbate 80™ and HCO-50), etc.Examples of the oily medium include sesame oil and soybean oil, whichmay also be used in combination with a dissolution aid such as benzylbenzoate and benzyl alcohol.

The prophylactic/therapeutic agent described above may further beformulated with a buffer (e.g., phosphate buffer, sodium acetatebuffer), a soothing agent (e.g., benzalkonium chloride, procainehydrochloride), a stabilizer (e.g., human serum albumin, polyethyleneglycol), a preservative (e.g., benzyl alcohol, phenol), an antioxidant,etc. The thus-prepared liquid for injection is normally filled in anappropriate ampoule.

Since the thus obtained pharmaceutical preparation is safe and lowtoxic, the preparation can be administered to a human or mammal (e.g.,rat, rabbit, sheep, pig, bovine, cat, dog, monkey).

The dose of the ligand protein of the present invention varies dependingon subject to be administered, organs to be administered, conditions,routes for administration, etc.; in oral administration, e.g., for apatient with cancer (as 60 kg body weight), the dose is normally about0.1 mg to about 100 mg, preferably about 1.0 to about 50 mg, and morepreferably about 1.0 to about 20 mg per day. In parenteraladministration, the single dose varies depending on subject to beadministered, target organ, conditions, routes for administration, etc.but it is advantageous, e.g., for a patient with cancer (as 60 kg bodyweight), to administer the active ingredient intravenously in a dailydose of about 0.01 to about 30 mg, preferably about 0.1 to about 20 mg,and more preferably about 0.1 to about 10 mg. For other animal species,the corresponding dose as converted per 60 kg body weight can beadministered.

The dose of the DNA encoding the ligand protein varies depending onsubject to be administered, organs to be administered, conditions,routes for administration, etc.; in oral administration, e.g., for apatient with cancer (as 60 kg body weight), the dose is normally about0.1 mg to about 100 mg, preferably about 1.0 to about 50 mg, and morepreferably about 1.0 to about 20 mg per day. In parenteraladministration, the single dose varies depending on subject to beadministered, target organ, conditions, routes for administration, etc.but it is advantageous, e.g., for a patient with cancer (as 60 kg bodyweight), to administer the active ingredient intravenously in a dailydose of about 0.01 to about 30 mg, preferably about 0.1 to about 20 mg,and more preferably about 0.1 to about 10 mg. For other animal species,the corresponding dose as converted per 60 kg body weight can beadministered.

<2> An Agent for Genetic Diagnosis

By using DNA encoding the ligand protein of the present invention or thereceptor protein as a probe, an aberration (genetic aberration) of theDNA or mRNA encoding the ligand protein of the present invention in ahuman or mammal (e.g., rat, rabbit, sheep, pig, bovine, cat, dog,monkey) can be detected. Therefore, the DNA of the present invention isuseful as an agent for genetic diagnosis for damage, mutation ordecreased expression of the DNA or mRNA, or increased expression oroverexpression of the DNA or mRNA.

The genetic diagnosis described above using the DNA encoding the ligandprotein of the present invention can be performed by, for example, thepublicly known Northern hybridization assay or the PCR-SSCP assay(Genomics, 5, 874-879 (1989); Proceedings of the National Academy ofSciences of the United States of America, 86, 2766-2770 (1989)).

<3> A Method of Quantifying the Receptor for the Ligand Protein of thePresent Invention

Since the receptor protein has binding affinity to the ligand of thepresent invention, the receptor concentration can be quantified in vivowith good sensitivity.

The quantification method of the present invention can be used incombination with, for example, a competitive method. The receptorconcentration in a test sample can be measured by contacting the testsample to the ligand protein of the present invention. Specifically, themethod can be used by following, for example, the methods described in(i) and (ii) below or modified methods thereof:

-   -   (i) Hiroshi Irie, ed. “Radioimmunoassay,” Kodansha, published in        1974    -   (ii) Hiroshi Irie, ed. “Sequel to the Radioimmunoassay,”        Kodansha, published in 1979.        <4> A Method of Screening a Compound (Agonist, Antagonist, or        the Like) that Alters the Binding Property Between the Ligand        Protein of the Present Invention and the Receptor

Using the receptor protein to the ligand protein of the presentinvention, or a salt thereof, or using the receptor binding assay systemof the expression system constructed using the recombinant receptorprotein, a compound (e.g., peptide, protein, non-peptide compound,synthetic compound, fermentation product) or a salt thereof that altersthe binding property between the ligand protein of the present inventionand the receptor protein can be efficiently screened. Said receptor usedherein includes one which is well identified in terms of properties, forexample, a G-protein coupled protein, and especially human, mouse or ratOT7T175 is preferred.

Such a compound includes (a) a compound that has the G protein-coupledreceptor-mediated cell-stimulating activities (e.g., activities ofpromoting or suppressing arachidonic acid release, acetylcholinerelease, intracellular Ca²⁺ release, intracellular cAMP production,intracellular cGMP production, inositol phosphate production, changes incell membrane potential, phosphorylation of intracellular proteins,activation of c-fos, pH reduction, etc.) (so-called an agonist to thereceptor protein); (b) a compound that does not have thecell-stimulating activity (so-called an antagonist to the receptorprotein); and (c) a compound that reduces the binding affinity betweenthe ligand protein and the receptor protein.

Thus, the present invention provides a method of screening a compound ora salt thereof that alters the binding property between the ligandprotein of the present invention and the receptor protein, whichcomprises comparing (i) the case wherein the ligand protein of thepresent invention is brought in contact with the receptor protein, with(ii) the case wherein the ligand protein is brought in contact with thereceptor and a test compound.

The screening method of the present invention is characterized byassaying, for example, the binding amount of the ligand to the receptor,the cell-stimulating activity, and comparing the assay results of (i)and (ii).

More specifically, the present invention provides the followingscreening methods:

(1) a method of screening a compound or a salt thereof that alters thebinding property between the ligand protein of the present invention andthe receptor protein, which comprises:

-   -   measuring the binding amount of the labeled ligand to the        receptor protein, when the labeled ligand is brought in contact        with the receptor protein and when the labeled ligand and a test        compound are brought in contact with the receptor protein, and,    -   comparing their binding amounts, (in this case, the labeled        receptor can replace the labeled ligand);

(2) a method of screening a compound or a salt that alters the bindingproperty between the ligand protein and the receptor protein, whichcomprises:

-   -   measuring the binding amount of the labeled ligand to a cell        containing the receptor protein or the membrane fraction        thereof, when the labeled ligand is brought in contact with the        cell or cell membrane fraction and when the labeled ligand and a        test compound are brought in contact with the cell or cell        membrane fraction, and    -   comparing their binding amounts;

(3) a method of screening a compound or a salt thereof that alters thebinding property between the ligand protein and the receptor protein,which comprises:

-   -   measuring the binding amount of the labeled ligand to the        receptor protein expressed on a cell membrane of a transformant        culture containing the DNA of the present invention, when the        labeled ligand is brought in contact with the receptor protein        expressed and when the labeled ligand and a test compound are        brought in contact with the receptor protein expressed, and,    -   comparing their binding amounts;

(4) a method of screening a compound or a salt thereof that alters thebinding property between the ligand protein and the receptor protein,which comprises:

-   -   measuring the receptor-mediated cell-stimulating activity (e.g.,        activity of promoting or suppressing arachidonic acid release,        acetylcholine release, intracellular Ca²⁺ release, intracellular        cAMP production, intracellular cGMP production, inositol        phosphate production, changes in cell membrane potential,        phosphorylation of intracellular proteins, activation of c-fos,        pH reduction, etc.), when the labeled ligand is brought in        contact with a cell containing the receptor protein and when the        labeled ligand and a test compound are brought in contact with        the cell, and,    -   comparing their activities; and

(5) a method of screening a compound or a salt thereof that alters thebinding property between the ligand protein and the receptor protein,which comprises:

-   -   measuring the receptor-mediated cell-stimulating activity (e.g.,        activity of promoting or suppressing arachidonic acid release,        acetylcholine release, intracellular Ca²⁺ release, intracellular        cAMP production, intracellular cGMP production, inositol        phosphate production, changes in cell membrane potential,        phosphorylation of intracellular proteins, activation of c-fos,        pH reduction, etc.), when the labeled ligand is brought in        contact with the receptor protein expressed on a cell membrane        of a transformant culture containing the DNA of the present        invention and when the labeled ligand and a test compound are        brought in contact with the receptor protein expressed, and,    -   comparing their activities.

Using any one of the methods (1) to (5), a compound that inhibits thebinding of the ligand and the receptor can be efficiently screened.Furthermore, it is easy to assess whether the obtained compound is anagonist or antagonist.

More specifically, the screening methods of the present invention aredescribed hereinafter.

First, for the receptor protein used for the screening methods of thepresent invention, any substance may be used so long as it contains thereceptor protein as described above. The cell membrane fraction frommammalian organs containing the receptor protein is preferred. However,it is preferable to use the receptor protein produced in a large amountusing a recombinant for the screening.

To produce the receptor protein, the methods described above are used,and it is preferred to express a DNA encoding the receptor protein inmammalian and insect cells. For the DNA fragment encoding the targetprotein, the complementary DNA, but not necessarily limited thereto, isemployed. For example, the gene fragments and synthetic DNA may also beused. To introduce a DNA fragment encoding the receptor protein intohost animal cells and efficiently express the DNA there, it is preferredto insert the DNA fragment downstream of a polyhedorin promoter ofnuclear polyhedrosis virus (NPV) belonging to baculovirus hosted byinsects, SV40-derived promoter, retrovirus promoter, metallothioneinpromoter, human heat shock promoter, cytomegalovirus promoter, or SRapromoter. The amount and quality of the expressed receptor are examinedby publicly known methods, for example, the method described in theliterature [Nambi, P. et al., The Journal of Biological Chemistry (J.Biol. Chem.), 267, 19555-19559, 1992].

Therefore, in the screening methods of the present invention, thematerial containing the receptor protein may be the receptor proteinpurified by publicly known methods, a cell containing the receptorprotein, or the cell membrane fraction containing the receptor protein.

When the cell containing the receptor protein is used for the screeningmethod of the present invention, the cell may be fixed withglutaraldehyde, formalin, etc. The fixation can be made by publiclyknown methods.

The cell containing the receptor protein refers to a host cell thatexpresses the receptor protein. For the host cell, Escherichia coli,Bacillus subtilis, yeast, insect cells, animal cells are preferred.

The cell membrane fraction refers to a fraction abundant in cellmembrane obtained by cell disruption and subsequent fractionation by apublicly known method. Useful cell disruption methods include cellsquashing using a Potter-Elvehjem homogenizer, disruption using a Waringblender or Polytron (produced by Kinematica Inc.), disruption byultrasonication, and disruption by cell spraying through thin nozzlesunder an increased pressure using a French press or the like. Cellmembrane fractionation is effected mainly by fractionation using acentrifugal force, such as centrifugation for fractionation and densitygradient centrifugation. For example, cell disruption fluid iscentrifuged at a low speed (500 rpm to 3,000 rpm) for a short period oftime (normally about 1 to about 10 minutes), the resulting supernatantis then centrifuged at a higher speed (15,000 rpm to 30,000 rpm)normally for 30 minutes to 2 hours. The precipitate thus obtained isused as the membrane fraction. The membrane fraction is rich in thereceptor protein expressed and membrane components such as cell-derivedphospholipids and membrane proteins.

The amount of the receptor protein in the cell or the membrane fractioncontaining the receptor protein is preferably 10³ to 10⁸ molecules percell, more preferably 10⁵ to 10⁷ molecules per cell. As the amount ofexpression increases, the ligand binding activity per unit of membranefraction (specific activity) increases so that not only the highlysensitive screening system can be constructed but also large quantity ofsamples can be assayed with the same lot.

To perform the screening methods (1) to (3) for a compound that altersthe binding property between the ligand protein of the present inventionand the receptor protein, for example, an appropriate receptor proteinfraction and the labeled ligand are necessary.

The receptor protein fraction is preferably a fraction of naturallyoccurring receptor protein or a recombinant receptor fraction having anactivity equivalent to that of the natural protein. Herein, theequivalent activity means a ligand binding activity, a signaltransduction activity or the like.

The labeled ligand includes a labeled ligand and a labeled ligandanalogue. For example, the ligand labeled with [³H], [¹²⁵I], [¹⁴C],[³⁵S], etc. are used (the labeled ligand protein of the presentinvention).

Specifically, for the screening for a compound that alters the bindingproperty between the ligand protein and the receptor protein, first, thereceptor protein standard is prepared by suspending the cell or cellmembrane fraction containing the receptor protein in a bufferappropriate for the screening. For the buffer, any buffer that does notinterfere with the binding of the ligand to the receptor is usable andexamples of such a buffer are phosphate buffer, Tris-hydrochloridebuffer, etc., having pH of 4 to 10 (preferably pH of 6 to 8). Tominimize a non-specific binding, a surfactant such as CHAPS, Tween-80™(Kao-Atlas Co.), digitonin, deoxycholate, etc. may be added to thebuffer. To inhibit degradation of the receptor and the ligand protein byproteases, protease inhibitors such as PMSF, leupeptin, E-64 (producedby Peptide Research Laboratory, Co.), and pepstatin may be added. To0.01 to 10 ml of the receptor solution, a given amount (5,000 to 500,000cpm) of the labeled ligand is added, and 10⁻⁴ M-10⁻¹⁰ M of a testcompound is simultaneously added thereto. To examine non-specificbinding (NSB), a reaction tube containing excess of the unlabeled ligandis also prepared. The reaction is carried out at approximately 0 to 50°C., preferably about 4 to 37° C. for about 20 minutes to about 24 hours,preferably about 30 minutes to about 3 hours. After completion of thereaction, the reaction mixture is filtrated through glass fiber filterpaper, etc. and washed with an appropriate volume of the same buffer.The residual radioactivity on the glass fiber filter paper is thenmeasured by means of a liquid scintillation counter or γ-counter.Regarding the count obtained by subtracting the amount of non-specificbinding (NSB) from the count obtained in the absence of any competitivesubstance (B₀) as 100%, when the amount of specific binding (B-NSB) is,for example, 50% or less, the test compound can be selected as acandidate substance having a potential of competitive inhibition.

To perform the screening methods (4) and (5) supra for a compound thatalters the binding property between the ligand protein and the receptorprotein, the receptor protein-mediated cell-stimulating activity (e.g.,activity of promoting or inhibiting arachidonic acid release,acetylcholine release, intracellular Ca²⁺ release, intracellular cAMPproduction, intracellular cGMP production, inositol phosphateproduction, changes in cell membrane potential, phosphorylation ofintracellular proteins, activation of c-fos, pH reduction, etc.) can bemeasured using publicly known methods or commercially available kits.

Specifically, the cell containing the receptor protein is first culturedon a multi-well plate, etc. Prior to screening, the medium is replacedwith fresh medium or with an appropriate non-cytotoxic buffer, followedby incubation for a given period of time in the presence of a testcompound. Subsequently, in the extract from the cells or the supernatantrecovered, any resulting product is quantified by appropriateprocedures. When it is difficult to detect the production of the indexsubstance (e.g., arachidonic acid) for the cell-stimulating activity,due to a degrading enzyme contained in the cells, an inhibitor againstsuch a degrading enzyme may be added prior to the assay. For detectingactivities such as the cAMP production suppression activity, thebaseline production in the cell is increased by forskolin or the likeand the suppressing effect on the increased baseline production may thenbe detected.

The screening by assaying the cell-stimulating activity requires a cellexpressing an appropriate receptor protein. For the cell expressing thereceptor protein, a cell line possessing the native receptor protein, acell line expressing the recombinant receptor protein described aboveand the like are desirable.

For a test compound, for example, a peptide, protein, non-peptidecompound, synthetic compound, fermentation product, cell extract, plantextract, and animal tissue extract are used. The compound may be novelor known.

The kit for screening a compound or a salt thereof that alters thebinding property between the ligand protein and the receptor proteincomprises the receptor protein, a cell containing the receptor protein,or a membrane fraction of the cell containing the receptor protein.

Examples of the screening kits of the present invention are as follow.

1. Reagents for the Screening

(i) Buffer for Measurement and Washing

Hanks' balanced salt solution (Gibco Co.) supplemented with 0.05% bovineserum albumin (Sigma Co.).

The solution is sterilized by filtration through a 0.45 m filter, andstored at 4° C. or may be prepared at use.

(ii) Standard G Protein-Coupled Receptor

CHO cells expressing the receptor protein are passaged in a 12-wellplate at a density of 5×10⁵ cells/well followed by culturing at 37° C.under 5% CO₂ and 95% air for 2 days.

(iii) Labeled Ligand

An aqueous solution of the ligand labeled with commercially available[³H], [¹²⁵I], [¹⁴C], [35S], etc. is stored at 4° C. or −20° C., anddiluted to 1 μM with the measurement buffer at use.

(iv) Standard Ligand Solution

The ligand protein of the present invention is dissolved in and adjustedto 1 mM with PBS containing 0.1% bovine serum albumin (Sigma Co.) andstored at −20° C.

2. Measurement Method

(i) CHO cells expressing the receptor protein are cultured in a 12-wellculture plate and washed twice with 1 ml of the measurement buffer, and490 μl of the measurement buffer is added to each well.

(ii) After adding 5 μl of 10⁻³-10⁻¹⁰ M test compound solution, 5 μl ofthe labeled ligand is added to the mixture, and the cells are incubatedat room temperature for an hour. To determine the amount of thenon-specific binding, 5 μl of the non-labeled ligand is added in placeof the test compound.

(iii) The reaction solution is removed, and the wells are washed 3 timeswith the washing buffer. The labeled ligand bound to the cells isdissolved in 0.2N NaOH-1% SDS, and mixed with 4 ml of liquidscintillator A (Wako Pure Chemical Industries, Ltd.)

(iv) The radioactivity is measured using a liquid scintillation counter(Beckman Co.), and the percent maximum binding (PMB) is calculated bythe equation below.PMB=[(B−NSB)/(B ₀ −NSB)]×100

-   -   PMB: Percent maximum binding    -   B: Value obtained in the presence of a test compound    -   NSB: Non-specific binding    -   B₀: Maximum binding

The compound or its salt, which is obtainable using the screening methodor the screening kit of the present invention, is one that alters thebinding property between the ligand protein of the present invention andthe receptor protein. Specifically, the compound is: (a) a compoundhaving the G protein-coupled receptor-mediated cell-stimulating activity(e.g., activity of promoting or inhibiting arachidonic acid release,acetylcholine release, intracellular Ca²⁺ release, intracellular cAMPproduction, intracellular cGMP production, inositol phosphateproduction, changes in cell membrane potential, phosphorylation ofintracellular proteins, activation of c-fos, pH reduction) (so-called anagonist to the receptor protein); (b) a compound having no cellstimulating-activity (so-called an antagonist to the receptor protein);or (c) a compound that reduces the binding affinity between the ligandprotein of the present invention and the receptor protein.

The compound may be a peptide, protein, non-peptide compound, syntheticcompound, fermentation product, and may be novel or known.

Since an agonist to the receptor protein has the same physiologicalactivity as that of the ligand of the present invention to the receptorprotein, it is useful as a safe and low-toxic pharmaceutical, dependingon the ligand activity.

Specifically, an agonist of the receptor protein has the activity ofinhibiting tumor metastasis, and thus is useful for the preventionand/or treatment of any type of cancers (e.g., cancers of lung, stomach,liver, pancreas, large intestine, rectum, colon, prostate, ovary,uterine cervix, breast, etc.)

Furthermore, an agonist of the receptor protein has the activity ofregulating placental function, and thus is useful for the preventionand/or treatment of choriocarcinoma, hydatid mole, invasive mole,miscarriage, fetal dysgenesis, dysbolism of saccharide, dysbolism oflipid, or induction of delivery.

Since an antagonist to the receptor protein can suppress thephysiological activities of the ligand protein to the receptor protein,it is useful as a safe and low-toxic pharmaceutical to inhibit theligand activity.

The compound that reduces the binding affinity between the ligandprotein of the present invention and the receptor protein is useful as asafe and low-toxic pharmaceutical to decrease the physiological activityof the ligand protein to the receptor receptor.

When a compound or its salt, which is obtainable using the screeningmethod or the screening kit of the present invention, is employed as apharmaceutical composition, the composition can be prepared in aconventional manner. For example, the compound can be formulated into atablet, capsule, elixir, microcapsule, aseptic solution, suspension,etc., as described for the pharmaceutical containing the DNA of thepresent invention.

The preparation thus obtained is safe and low-toxic, and can beadministered to, for example, a human and mammal (e.g., rat, rabbit,sheep, pig, bovine, cat, dog, monkey).

The dose of the compound or its salt varies depending on subject to beadministered, target organs, conditions, routes for administration, etc.In oral administration, e.g., to a patient with cancer (as 60 kg bodyweight), the dose is normally about 0.1 mg to about 100 mg, preferablyabout 1.0 to about 50 mg, and more preferably about 1.0 to about 20 mgper day. In parenteral administration, the single dose varies dependingon a subject to be administered, target organ, conditions, routes foradministration, etc. but it is advantageous, e.g., for a patient withcancer (as 60 kg body weight), to administer the active ingredientintravenously in a daily dose of about 0.01 to about 30 mg, preferablyabout 0.1 to about 20 mg, and more preferably about 0.1 to about 10 mg.For other animal species, the corresponding dose as converted per 60 kgbody weight can be administered.

<5> A Prophylactic and/or Therapeutic Agent for Various DiseasesComprising a Compound (Agonist or Antagonist) that Alters the BindingProperty Between the Ligand Protein of the Present Invention and theReceptor

As described above, the ligand protein of the present invention has theactivity of inhibiting tumor metastasis, and thus is useful for theprevention and/or treatment of any type of cancers (e.g., cancers oflung, stomach, liver, pancreas, large intestine, rectum, colon,prostate, ovary, uterine cervix, breast, etc.)

Furthermore, an agonist of the receptor protein has the activity ofregulating placental function, and thus is useful for the preventionand/or treatment of choriocarcinoma, hydatid mole, invasive mole,miscarriage, fetal dysgenesis, dysbolism of saccharide, dysbolism oflipid, or induction of delivery.

Therefore, the compound (agonist or antagonist) that alters the bindingproperty between the ligand protein of the present invention and thereceptor can be used as a prophylactic and/or therapeutic agent fordiseases associated with dysfunction, deficiency or excess of the ligandprotein of the present invention.

When the compound is used as the prophylactic and/or therapeutic agentfor diseases associated with dysfunction, deficiency or excess of theligand protein of the present invention, the formulation can be made ina conventional manner.

For example, the compound can be administered orally as sugar coatedtablet, capsule, elixir, and microcapsule, or non-orally as injectionsuch as aseptic solution or suspension in water or otherpharmaceutically acceptable liquid. For example, these formulations canbe produced by mixing the compound with a physiologically acceptableknown carrier, flavor, filler, vehicle, antiseptic, stabilizer, andbinder in a unit-dosage form generally approved for drug preparation.The amount of the effective ingredient is set to an appropriate amountwithin the specified range.

An additive that may be mixed in a tablet, capsule, etc. includes, forexample, binders such as gelatin, cornstarch, tragacanth, and acacia,fillers such as crystalline cellulose, imbibers such as cornstarch,gelatin, and alginic acid, lubricants such as magnesium stearate,sweeteners such as sucrose and saccharin, and flavors such aspeppermint, akamono oil and cherry. When the dosage form is a capsule,liquid carrier such as fat and oil may be contained. An asepticcomposition for injection can be prepared following the conventionaltechnique for the preparation, such as by dissolving or suspending theactive substance in a vehicle, e.g., water for injection, and naturalplant oil e.g., sesame oil and coconut oil. For the aqueous solution forinjection, for example, a physiological saline and isotonic solution(e.g., D-sorbitol, D-mannitol, sodium hydrochloride) containing glucoseand other adjuvant are used. An appropriate dissolution-assisting agent,for example, alcohol (e.g., ethanol), polyalcohol (e.g., propyleneglycol, polyethylene glycol), nonionic surfactant (e.g., polysorbate 8™,HCO-50) may be combined. For the oily solution, for example, sesame oiland soybean oil are used, and dissolution-assisting agents such asbenzyl benzoate and benzyl alcohol may be combined.

The prophylactic/therapeutic agent as described above may be combined,for example, with a buffer (e.g., phosphate buffer, sodium acetatebuffer), soothing agent (e.g., benzalkonium chloride, procainehydrochloride), stabilizer (e.g., human serum albumin, polyethyleneglycol), preservative (e.g., benzyl alcohol, phenol), and antioxidant.The prepared injection solution is usually filled in an appropriateampoule.

The preparation thus obtained is safe and low toxic, and can beadministered to, for example, a human and mammal (e.g., rat, rabbit,sheep, pig, bovine, cat, dog, monkey).

The dose of the compound or its salt varies depending on the subject tobe administered, target organs, conditions, routes for administration,etc. In oral administration, e.g., to a patient with cancer (as 60 kgbody weight), the dose is normally about 0.1 mg to about 100 mg,preferably about 1.0 to about 50 mg, and more preferably about 1.0 toabout 20 mg per day. In parenteral administration, the single dosevaries depending on the subject to be administered, target organ,conditions, routes for administration, etc. but it is advantageous,e.g., for a patient with cancer (as 60 kg body weight), to administerthe active ingredient intravenously in a daily dose of about 0.01 toabout 30 mg, preferably about 0.1 to about 20 mg, and more preferablyabout 0.1 to about 10 mg. For other animal species, the correspondingdose as converted per 60 kg body weight can be administered.

<6> Quantification of the Ligand Protein of the Present Invention

The antibody to ligand protein or receptor protein of the presentinvention is capable of specifically recognizing the ligand protein orreceptor protein of the present invention. Therefore, the antibody canbe used to quantify the ligand protein or receptor protein of thepresent invention in a test fluid, especially for quantification by thesandwich immunoassay. Thus, the present invention provides, for example,the following quantification methods:

-   -   (i) a method of quantifying the ligand protein or receptor        protein of the present invention in a test fluid, which        comprises competitively reacting the antibody to the ligand        protein or receptor protein with the test fluid and a labeled        form of the ligand protein or receptor protein, and measuring        the ratio of the labeled ligand protein or receptor protein        bound to the antibody; and    -   (ii) a method of quantifying the ligand protein or receptor        protein of the present invention in a test fluid, which        comprises reacting the test fluid with the antibody to the        ligand protein or receptor protein immobilized on a carrier and        a labeled form of the antibody simultaneously or sequentially,        and measuring the activity of the label on the immobilized        carrier.

In (ii) described above, it is preferred that one antibody recognizesthe N-terminal region of the ligand protein or receptor protein of thepresent invention, and another antibody reacts with the C-terminalregion of the ligand protein or receptor protein.

Using a monoclonal antibody to the ligand protein or receptor protein ofthe present invention (hereinafter sometimes referred to as themonoclonal antibody of the present invention), the ligand protein orreceptor protein of the present invention can be assayed and alsodetected by tissue staining or the like. For this purpose, the antibodymolecule itself may be used, or F(ab′)₂, Fab′ or Fab fractions of theantibody molecule may also be used. The type of assay method using theantibody to the ligand protein or receptor protein of the presentinvention is not particularly limited. Any assay method can be used, solong as the amount of antibody, antigen, or antibody-antigen complex,corresponding to the amount of antigen (e.g., the amount of the receptorprotein) in the test fluid, can be detected by chemical or physicalmeans, and then the amount of the antigen can be calculated from astandard curve prepared from standard solutions containing known amountsof the antigen. For example, nephrometry, competitive methods,immunometric method, and sandwich method are appropriately used, withthe sandwich method described below being most preferable in terms ofsensitivity and specificity.

As the labeling agent for the methods using labeled substances, thereare employed, for example, radioisotopes, enzymes, fluorescentsubstances, luminescent substances, etc. For the radioisotope, forexample, [125I], [¹³¹I], [³H] and [¹⁴C] are used. As the enzymedescribed above, stable enzymes with high specific activity arepreferred; for example, β-galactosidase, β-glucosidase, alkalinephosphatase, peroxidase, malate dehydrogenase and the like are used.Example of the fluorescent substance used are fluorescamine andfluorescein isothiocyanate are used. For the luminescent substance, forexample, luminol, luminol derivatives, luciferin, and lucigenin.Furthermore, the biotin-avidin system may be used for binding antibodyor antigen to the label.

For immobilization of antigen or antibody, physical adsorption may beused. Chemical binding methods conventionally used for insolubilizationor immobilization of proteins or enzymes may also be used. For thecarrier, for example, insoluble polysaccharides such as agarose,dextran, cellulose, etc.; synthetic resin such as polystyrene,polyacrylamide, silicon, etc., and glass or the like are used.

In the sandwich method, the immobilized monoclonal antibody of thepresent invention is reacted with a test fluid (primary reaction), thenwith the labeled monoclonal antibody of the present invention (secondaryreaction), and the activity of the label on the immobilizing carrier ismeasured, whereby the amount of the ligand protein or receptor proteinof the present invention in the test fluid can be quantified. The orderof the primary and secondary reactions may be reversed, and thereactions may be performed simultaneously or with an interval. Themethods of labeling and immobilization can be performed by the methodsdescribed above.

In the immunoassay by the sandwich method, the antibody used forimmobilized or labeled antibodies is not necessarily one species, but amixture of two or more species of antibody may be used to increase themeasurement sensitivity.

In the methods of assaying the ligand protein or receptor protein of thepresent invention by the sandwich method, antibodies that bind todifferent sites of the ligand protein are preferably used as themonoclonal antibodies of the present invention for the primary andsecondary reactions. That is, in the antibodies used for the primary andsecondary reactions are, for example, when the antibody used in thesecondary reaction recognizes the C-terminal region of the receptorprotein, it is preferable to use the antibody recognizing the regionother than the C-terminal region for the primary reaction, e.g., theantibody recognizing the N-terminal region.

The monoclonal antibody to the ligand protein or receptor protein of thepresent invention can be used for the assay systems other than thesandwich method, for example, competitive method, immunometric method,nephrometry, etc. In the competitive method, antigen in a test fluid andthe labeled antigen are competitively reacted with antibody, and theunreacted labeled antigen (F) and the labeled antigen bound to theantibody (B) are separated (B/F separation). The amount of the label inB or F is measured, and the amount of the antigen in the test fluid isquantified. This reaction method includes a liquid phase method using asoluble antibody as an antibody, polyethylene glycol for B/F separationand a secondary antibody to the soluble antibody, and an immobilizedmethod either using an immobilized antibody as the primary antibody, orusing a soluble antibody as the primary antibody and immobilizedantibody as the secondary antibody.

In the immunometric method, antigen in a test fluid and immobilizedantigen are competitively reacted with a definite amount of labeledantibody, the immobilized phase is separated from the liquid phase, orantigen in a test fluid and an excess amount of labeled antibody arereacted, immobilized antigen is then added to bind the unreacted labeledantibody to the immobilized phase, and the immobilized phase isseparated from the liquid phase. Then, the amount of the label in eitherphase is measured to quantify the antigen in the test fluid.

In the nephrometry, insoluble precipitate produced after theantigen-antibody reaction in gel or solution is quantified. When theamount of antigen in the test fluid is small and only a small amount ofprecipitate is obtained, laser nephrometry using scattering of laser isadvantageously employed.

For applying these immunological methods to the measurement methods ofthe present invention, any particular conditions or procedures are notrequired. Systems for measuring the ligand protein or receptor proteinof the present invention are constructed by adding the usual technicalconsideration in the art to the conventional conditions and procedures.For the details of these general technical means, reference can be madeto the following reviews and texts. [For example, Hiroshi Irie, ed.“Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie, ed.“Sequel to the Radioimmunoassay” (Kodansha, published in 1979), EijiIshikawa, et al. ed. “Enzyme immonoassay” (Igakushoin, published in1978), Eiji Ishikawa, et al. ed. “Immunoenzyme assay” (2nd ed.)(Igakushoin, published in 1982), Eiji Ishikawa, et al. ed. “Immunoenzymeassay” (3rd ed.) (Igakushoin, published in 1987), Methods in ENZYMOLOGY,Vol. 70 (Immunochemical Techniques (Part A)), ibid., Vol. 73(Immunochemical Techniques (Part B)), ibid., Vol. 74 (ImmunochemicalTechniques (Part C)), ibid., Vol. 84 (Immunochemical Techniques (Part D:Selected Immunoassays)), ibid., Vol. 92 (Immunochemical Techniques (PartE: Monoclonal Antibodies and General Immunoassay Methods)), ibid., Vol.121 (Immunochemical Techniques (Part I: Hybridoma Technology andMonoclonal Antibodies))(all published by Academic Press Publishing).

As described above, the ligand protein or receptor protein of thepresent invention can be quantified with high sensitivity, using theantibody of the present invention.

By quantifying the ligand protein or receptor protein of the presentinvention in vivo using the antibody of the present invention, diagnosiscan be made on various diseases associated with dysfunction of theligand protein of the present invention.

The antibody to the ligand protein or receptor protein of the presentinvention can also be used for specifically detecting the ligand proteinor receptor protein of the present invention present in test samplessuch as body fluids or tissues. The antibody may also be used forpreparation of antibody columns for purification of the ligand proteinor receptor protein of the present invention, for detection of theligand protein of the present invention in each fraction uponpurification, and for analysis of the behavior of the ligand protein orreceptor protein of the present invention in the test cells.

<7> Neutralization with the Antibody to the Ligand Protein of thePresent Invention

The neutralizing activity of the antibody to the ligand protein orreceptor protein of the present invention refers to an activity ofinactivating the signal transduction function involving the ligandprotein or receptor protein. Therefore, when the antibody has theneutralizing activity, the antibody can inactivate the signaltransduction involving the ligand protein or receptor protein, forexample, inactivate the ligand protein-mediated cell-stimulatingactivity (e.g., activity of promoting or inhibiting arachidonic acidrelease, acetylcholine release, intracellular Ca²⁺ release,intracellular cAMP production, intracellular cGMP production, inositolphosphate production, changes in cell membrane potential,phosphorylation of intracellular proteins, activation of c-fos, decreasein pH, etc.). Therefore, the antibody can be used for the preventionand/or treatment of diseases caused by overexpression of the ligandprotein or receptor protein.

<8> Preparation of Animals Carrying the DNA Encoding the Ligand Proteinof the Present Invention

Using the DNA of the present invention, transgenic animals expressingthe ligand protein of the present invention can be prepared. Examples ofthe animals include mammals (e.g., rats, mice, rabbits, sheep, swine,bovine, cats, dogs, monkeys, etc.) can be used, with mice and rabbitsbeing particularly appropriate.

To transfer the DNA of the present invention to target animals, it isgenerally advantageous to use a gene construct in which the DNA isligated downstream of a promoter suitable for expression in animalcells. For example, when the DNA of the present invention derived from amouse is transferred, a gene construct in which the DNA is ligateddownstream of a promoter, which can express DNAs of animals highlyhomologous to the DNA, is microinjected to, for example, a mousefertilized egg; thus, the DNA-transferred animal, which is capable ofproducing a high level of the receptor protein of the present invention,can be produced. Examples of the promoters that are usable includevirus-derived promoters and ubiquitous expression promoters such asmetallothionein promoter, but promoters of NGF gene and enolase that arespecifically expressed in the brain are preferably used.

The transfer of the DNA of the present invention at the fertilized eggcell stage secures the presence of the DNA in all germ and somatic cellsin the produced animal. The presence of the ligand protein of thepresent invention in the germ cells in the DNA-transferred animal meansthat all germ and somatic cells contain the ligand protein of thepresent invention in all progenies of the animal. The progenies of theanimal that took over the gene contain the ligand protein of the presentinvention in all germ and somatic cells.

The DNA-transferred animals of the present invention can be maintainedand bled in the conventional environment as animals carrying the DNAafter confirming the stable retention of the gene in the animals throughmating. Furthermore, mating male and female animals containing theobjective DNA results in acquiring homozygote animals having thetransferred gene on both homologous chromosomes. By mating the male andfemale homozygotes, bleeding can be performed so that all progeniescontain the DNA.

Since the ligand protein of the present invention is highly expressed inthe animals in which the DNA of the present invention has beentransferred, the animals are useful for screening of agonists orantagonists to the ligand protein of the present invention.

The animals in which the DNA of the present invention has beentransferred can also be used as cell sources for tissue culture. Theligand protein of the present invention can be analyzed by, for example,directly analyzing the DNA or RNA in tissues from the mouse in which theDNA of the present invention has been transferred, or by analyzingtissues containing the ligand protein expressed from the gene. Cellsfrom tissues containing the ligand protein of the present invention arecultured by the standard tissue culture technique. Using these cells,for example, the function of tissue cells such as cells derived from thebrain or peripheral tissues, which are generally difficult to culture,can be studied. Using these cells, for example, it is possible to selectpharmaceuticals that increase various tissue functions. When a highlyexpressing cell line is available, the ligand protein of the presentinvention can be isolated and purified from the cell line.

In the specification and drawings, the codes of bases and amino acidsare denoted in accordance with the IUPAC-IUB Commission on BiochemicalNomenclature or by the common codes in the art, examples of which areshown below. For amino acids that may have the optical isomer, L form ispresented unless otherwise indicated.

-   -   DNA: deoxyribonucleic acid    -   cDNA: complementary deoxyribonucleic acid    -   A: adenine    -   T: thymine    -   G: guanine    -   C: cytosine    -   RNA: ribonucleic acid    -   mRNA: messenger ribonucleic acid    -   dATP: deoxyadenosine triphosphate    -   dTTP: deoxythymidine triphosphate    -   dGTP: deoxyguanosine triphosphate    -   dCTP: deoxycytidine triphosphate    -   ATP: adenosine triphosphate    -   EDTA: ethylenediaminetetraacetic acid    -   SDS: sodium dodecyl sulfate    -   Gly: glycine    -   Ala: alanine    -   Val: valine    -   Leu: leucine    -   Ile: isoleucine    -   Ser: serine    -   Thr: threonine    -   Cys: cysteine    -   Met: methionine    -   Glu: glutamic acid    -   Asp: aspartic acid    -   Lys: lysine    -   Arg: arginine    -   His: histidine    -   Phe: phenylalanine    -   Tyr: tyrosine    -   Trp: tryptophan    -   Pro: proline    -   Asn: asparagine    -   Gln: glutamine    -   pGlu: pyroglutamic acid    -   Me: methyl    -   Et: ethyl    -   Bu: butyl    -   Ph: phenyl    -   TC: thiazolidine-4(R)-carboxamide

The substituents, protective groups and reagents, which are frequentlyused throughout the specification, are shown by the followingabbreviations.

-   -   Tos: p-toluenesulfonyl    -   CHO: formyl    -   Bzl: benzyl    -   Cl₂Bl: 2,6-dichlorobenzyl    -   Bom: benzyloxymethyl    -   Z: benzyloxycarbonyl    -   Cl-Z: 2-chlorobenzyloxycarbonyl    -   Br-Z: 2-bromobenzyloxycarbonyl    -   Boc: t-butoxycarbonyl    -   DNP: dinitrophenol    -   Trt: trityl    -   Bum: t-butoxymethyl    -   Fmoc: N-9-fluorenylmethoxycarbonyl    -   HOBt: 1-hydroxybenztriazole    -   HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine    -   HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide    -   DCC: N,N′-dicyclohexylcarbodiimide    -   BHA: benzhydrylamine    -   MeBzl: 4-methylbenzyl    -   OcHex: cyclohexylester    -   NMP: N-methylpyrrolidone    -   TFA: trifluoroacetic acid

The sequence identification numbers (SEQ ID NO) in the sequence listingof the specification indicates the following sequence, respectively.

[SEQ ID NO:1]

This shows the amino acid sequence of a novel mouse ligand proteinKiSS-1 of the present invention (mouse type 1).

[SEQ ID NO:2]

This shows the amino acid sequence of a novel mouse ligand proteinKiSS-1 of the present invention (mouse type 2).

[SEQ ID NO:3]

This shows the amino acid sequence of a novel rat ligand protein KiSS-1of the present invention (rat type).

[SEQ ID NO:4]

This shows the cDNA sequence encoding a novel mouse ligand proteinKiSS-1 of the present invention (mouse type 1).

[SEQ ID NO:5]

This shows the cDNA sequence encoding a novel mouse ligand proteinKiSS-1 of the present invention (mouse type 2).

[SEQ ID NO:6]

This shows the cDNA sequence encoding a novel rat ligand protein KiSS-1of the present invention (rat type).

[SEQ ID NO:7]

This shows the amino acid sequence of a novel human G protein-coupledreceptor protein hOT7T175.

[SEQ ID NO:8]

This shows the amino acid sequence of a novel rat G protein-coupledreceptor protein rOT7T175.

[SEQ ID NO:9]

This shows the nucleic acid sequence of primer A used for cloning amouse KiSS-1 of the present invention.

[SEQ ID NO:10]

This shows the nucleic acid sequence of primer B used for cloning amouse KiSS-1 of the present invention.

[SEQ ID NO:11]

This shows the nucleic acid sequence of primer C used for cloning amouse KiSS-1 of the present invention.

(SEQ ID NO:12]

This shows the nucleic acid sequence of primer D used for cloning amouse KiSS-1 of the present invention.

[SEQ ID NO:13]

This shows the nucleic acid sequence of primer E used for cloning amouse KiSS-1 of the present invention.

[SEQ ID NO:14]

This shows the nucleic acid sequence of a degenerate primer 13-3F38 usedfor cloning cDNA encoding the rat ligand (1-54) [rat KiSS-1] of thepresent invention.

[SEQ ID NO:15]

This shows the nucleic acid sequence of a degenerate primer KiSS357Rused for cloning cDNA encoding the rat ligand (1-54) [rat KiSS-1] of thepresent invention.

[SEQ ID NO:16]

This shows the nucleic acid sequence of DNA fragment encoding a partialpeptide of rat KiSS-1.

[SEQ ID NO:17]

This shows the nucleic acid sequence of primer 288-41F for obtaining DNAfragment encoding the entire peptide of rat KiSS-1.

[SEQ ID NO:18]

This shows the nucleic acid sequence of primer 288-10F for obtaining DNAfragment encoding the entire peptide of rat KiSS-1.

[SEQ ID NO:19]

This shows the nucleic acid sequence of primer 288-254R for obtainingDNA fragment encoding the entire peptide of rat KiSS-1.

[SEQ ID NO:20]

This shows the nucleic acid sequence of primer 288-44R for obtaining DNAfragment encoding the entire peptide of rat KiSS-1.

[SEQ ID NO:21]

This shows the nucleic acid sequence of primer rKiSS364F for obtainingDNA fragment encoding the entire peptide of rat KiSS-1.

[SEQ ID NO:22]

This shows the nucleic acid sequence of primer rKiSS859R for obtainingDNA fragment encoding the entire peptide of rat KiSS-1.

[SEQ ID NO:23]

This shows the nucleic acid sequence of 393 bp DNA fragment encoding theentire peptide of rat KiSS-1.

[SEQ ID NO:24]

This shows the amino acid sequence of a novel mouse G protein-coupledreceptor protein mOT7T175.

[SEQ ID NO:25]

This shows the DNA sequence encoding a novel mouse G protein-coupledreceptor protein mOT7T175.

[SEQ ID NO:26]

This shows the nucleic acid sequence of primer 1 used in Example 3.

[SEQ ID NO:27]

This shows the nucleic acid sequence of primer 2 used in Example 3.

Escherichia coli transformant DH10B/pCMV-mKiSS-1 obtained in Example 1was on deposit with the Ministry of Economy, Trade and Industry, Agencyof Industrial Science and Technology, National Institute of Bioscienceand Human Technology (NIBH), located at 1-1-3, Higashi, Tsukuba-shi,Ibaraki, Japan, as the Accession Number FERM BP-7003 on Jan. 24, 2000and with Institute for Fermentation (IFO), located at 2-17-85, JusoHoncho, Yodogawa-ku, Osaka-shi, Osaka, Japan, as the Accession NumberIFO 16348 on Dec. 16, 1999.

Escherichia coli transformant DH5α/pCR2.1-mKiSS-1.4A obtained in Example1 was on deposit with the Ministry of Economy, Trade and Industry,Agency of Industrial Science and Technology, National Institute ofBioscience and Human Technology (NIBH), located at 1-1-3, Higashi,Tsukuba-shi, Ibaraki, Japan, as the Accession Number FERM BP-7073 onMar. 6, 2000 and with Institute for Fermentation (IFO), located at2-17-85, Juso Honcho, Yodogawa-ku, Osaka-shi, Osaka, Japan, as theAccession Number IFO 16360 on Feb. 16, 2000.

Escherichia coli transformant TOP10/pRKISS4 obtained in Example 2 was ondeposit with the Ministry of Economy, Trade and Industry, Agency ofIndustrial Science and Technology, National Institute of Bioscience andHuman Technology (NIBH), located at 1-1-3, Higashi, Tsukuba-shi,Ibaraki, Japan, as the Accession Number FERM BP-7093 on Mar. 16, 2000and with Institute for Fermentation (IFO), located at 2-17-85, JusoHoncho, Yodogawa-ku, Osaka-shi, Osaka, Japan, as the Accession NumberIFO 16362 on Feb. 2, 2000.

Escherichia coli transformant DH5a/pCR-BluntII-mOT7T175 obtained inExample 3 was on deposit with the Ministry of Economy, Trade andIndustry, Agency of Industrial Science and Technology, NationalInstitute of Bioscience and Human Technology (NIBH), located at 1-1-3,Higashi, Tsukuba-shi, Ibaraki, Japan, as the Accession Number FERMBP-7428 on Jan. 11, 2001 and with Institute for Fermentation (IFO),located at 2-17-85, Juso Honcho, Yodogawa-ku, Osaka-shi, Osaka, Japan,as the Accession Number IFO 16523 on Dec. 22, 2000.

The present invention is described in detail below with reference toExamples, but is not deemed to limit the scope of the present inventionthereto. The gene manipulation procedures using Escherichia coli wereperformed as described in the Molecular Cloning.

EXAMPLE 1

Cloning of Mouse KiSS-1

cDNA cloning was performed according to the manufacturer's instructionof GENE TRAPPER (Life Technologies). Probe A (SEQ ID NO: 9) wasbiotinylated and then hybridized with single-stranded mouse embryo cDNAlibrary (Superscript cDNA library, Life Technologies) to obtain asingle-stranded gene, which was then converted to double-stranded geneusing primer B (SEQ ID NO. 10). This gene was introduced intoEscherichia coli DH108 by electroporation, and the transformants wereselected for ampicillin-resistance. Electroporation was performed usingE. coli Pulser (BIO RAD) at a voltage of 1.8 kV. Transformants thusobtained were subjected to screening by colony PCR using primer B (SEQID NO. 10) and primer C (SEQ ID NO. 11), and a transformant (Escherichiacoli) DH108/pCMV-mKiSS-1 was obtained. The ORF (open reading frame) (SEQID NO. 4) was deduced from the nucleotide sequence of this cDNA clone,and a novel secretory protein having the amino acid sequence (SEQ IDNO. 1) was designated mKiSS-1 (mouse type I).

For colony PCR, the reaction was carried out in the volume of 10 μlcontaining 1/50 volume of Advantage2 cDNA polymerase Mix (CLONTECH), 0.2μM each of primer B (SEQ ID NO. 10) and primer C (SEQ ID NO. 11), 200 μMdNTPs, 1/25 volume of DMSO and the buffer attached to the enzymeproduct. The PCR was carried out (i) for 10 minutes at 94° C., followedby (ii) 25 cycles of 94° C. for 10 seconds, 60° C. for 10 seconds and68° C. for 1 minutes.

Then, primers D (SEQ ID NO. 12) and E (SEQ ID NO. 13), corresponding tothe outside sequences of the ORF, were generated based on the abovesequence of pCMV-mKiSS-1, and PCR was performed using mouse embryoMarathon Ready cDNA (CLONETECH) as a template. The PCR was performed inthe reaction volume of 25 μl containing 1/50 volume of Advantage2 cDNApolymerase Mix (CLONTECH), 0.2 μM each of primer D (SEQ ID NO. 12) andprimer E (SEQ ID NO. 13), 200 μM dNTPs, 1/25 volume of DMSO and thebuffer attached to the enzyme product, with the following conditions:(i) for 2 minutes at 94° C., followed by (ii) 3 cycles of 94° C. for 10seconds and 68° C. for 1 minute and 30 seconds, (iii) 3 cycles of 94° C.for 10 seconds, 64° C. for 10 seconds and 68° C. for 1 minute, then (iv)30 cycles of 94° C. for 10 seconds and 60° C. for 10 seconds and 68° C.for 1 minute, and finally (v) 68° C. for 8 minutes for extension. Theproducts obtained from the PCR were subcloned into plasmid vectorpCR2.1-TOPO (Invitrogen), according to the manufacturer's instruction ofthe TOPO-TA cloning kit (Invitrogen), and then introduced into E. coliDH5a. The clones harboring the cDNA were selected on LB-agar mediumcontaining ampicillin. Analysis of the sequence of each clone revealedthat two types of clones were obtained: (i) a clone, nucleotide sequenceof which is completely identical to that of mKiSS-1 described above (SEQID NO. 4), and (ii) a clone having 12 nucleotide insertion in thenucleotide sequence of mKiSS-1 described above (SEQ ID NO. 4) andone-nucleotide mutation (no amino acid change) at nucleotide position402 from the starting position of the ORF. The 12-nucleotide insertionof (ii) can be translated into four amino acids, and both human KiSS-1and rat KiSS-1 contain an amino acid sequence corresponding to thesequence of the four amino acids. Therefore, we considered that a mousehas two types of mKiSS-1, and designated the novel secretory proteinhaving the amino acid sequence (SEQ ID NO. 2) deduced from the newlyobtained nucleotide sequence (SEQ ID NO. 5) as mKiSS-1-4A (mouse type2). Escherichia coli was transformed with the clone having thenucleotide sequence to obtain a transformant: Escherichia coliDH5a/pCR2.1-mKiSS-1.4A.

Amino acid sequences of mouse type 1 and type 2 KiSS-1 proteins (SEQ IDNOs: 4 and 5, respectively) deduced from the nucleotide sequences thusobtained showed high homology to the known human homolog (see FIG. 1).Probe A 5′-tat ggg gag ccg ctg gca aaa gtg-3′ Primer B 5′-tag acc tgcccc ttc ctc cca ga-3′ Primer C 5′-ctg ctg gcc tgt gga tcc agg ctt-3′Primer D 5′-tgc agg aga gtg aag att aaa tcc cca-3′ Primer E 5′-gag gacctg tcc cat ctc gca gga gtc a-3′

EXAMPLE 2

Cloning of cDNA that Encodes Rat Ligand (1-54) [Rat KiSS-1]

Total RNA was extracted from rat placenta using TRIZOL reagent (GibcoBRL) according to the method described in the manufacturer'sinstruction. Next, poly(A)⁺RNA was prepared from the total RNA usingolig-dT cellulose column (MessageMaker reagent assembly, Gibco BRL) inaccordance with the method described in the manufacturer's instruction.Subsequently, first strand cDNA was synthesized from the poly(A)+ RNAusing SuperScript Preamplification System for First Strand cDNASynthesis (Gibco BRL) in accordance with the method described in themanufacturer's instruction. The degenerate primers as shown below weredesigned based on the mouse KiSS-1 sequence and synthesized: 13-3F38:(SEQ ID NO. 14) 5′-TTCTTGGCAGCTRCTGCTTYTCCTCTGTG-3′ KiSS357R: (SEQ IDNO. 15) 5′-GAAGCGCAGGCCGAAGGAGTTCCA-3′

Degenerative PCR was performed using the above-mentioned first strandcDNA as a template, and 13-3F38 and KiSS357R as primers. The reactionsolution for the PCR was prepared by mixing 1 μl of Taq polymerase(TAKARA SHUZO), 10 μl of 10×PCR buffer (500 mM KCl, 100 mM Tris.HCl, pH8.3), 6 μl of 25 mM MgCl₂, 8 μl of 2.5 mM dNTP mixture, 4 μl of 25% DMSOsolution, 2 μl each of primers 13-3F38 and KiSS357R (20 μM each), 2 μlof the template cDNA (the first strand cDNA described above) and 65 μlof distilled water. The PCR was performed with the following conditionsto obtain PCR products: (1) initial denaturation (94° C. for 5 minutes),(2) cycle reaction (94° C. for 20 seconds-72° C. for 50 seconds), (3)cycle reaction (94° C. for 20 seconds-71.5° C. for 20 seconds-72° C. for30 seconds), (4) cycle reaction (94° C. for 20 seconds-71° C. for 20seconds-72° C. for 30 seconds), (5) cycle reaction (94° C. for 20seconds-70.5° C. for 20 seconds-72° C. for 30 seconds), (6) cyclereaction (94° C. for 20 seconds-72° C. for 20 seconds-72° C. for 30seconds), (7) cycle reaction (94° C. for 20 seconds-69.5° C. for 20seconds-72° C. for 30 seconds), (8) cycle reaction (94° C. for 20seconds-69° C. for 20 seconds-72° C. for 30 seconds), (9) cycle reaction(94° C. for 20 seconds-68.5° C. for 20 seconds-72° C. for 30 seconds),(10) cycle reaction (94° C. for 20 seconds-68° C. for 20 seconds-72° C.for 30 seconds), (11) 30-cycle reaction (94° C. for 20 seconds-61.8° C.for 20 seconds-72° C. for 30 seconds), followed by (12) final extension(72° C. for 7 minutes).

The PCR products were then electrophoresed on 1.5% agarose gel, and agel piece containing a cyber green-stained band of about 300 bp was cutout. A DNA fragment as the PCR product was recovered from the gel pieceusing the Gene Clean DNA extraction kit (BIO 101). Reaction fordetermination of nucleotide sequence of the DNA fragment was conductedusing the Dye Terminator Cycle Sequencing Kit (Applied Biosystems,Perkin-Elmer), and the nucleotide sequence of the PCR product wasdetermined using the automated fluorescence sequencer (DNA sequencerPrism 377: Applied Biosystems, Perkin-Elmer). As a result, the DNAfragment having the sequence encoding a partial peptide of rat KiSS-1was obtained. The following sequence is a nucleotide sequence of pCR288bp (SEQ ID NO. 16) consisting of 288 bp without the degenerate primerportion. pCR288bp: (SEQ ID NO. 16) TGGCCTCTTT TGGGGAGCCA CTGGCAAAAATGGCACCTGT GGTGAACCCT GAACCCACAG  60 GCCAACAGTC CGGACCCCAG GAACTCGTTAATGCCTGGCA AAAGGGCCCG CGGTATGCAG 120 AGAGCAAGCC TGGGGCTGCA GGACTGCGCGCTCGCCGAAC ATCGCCATGC CCGCCGGTGG 180 AGAACCCCAC GGGGCACCAG CGGCCCCCGTGTGCCACCCG CAGTCGCCTG ATCCCTGCGC 240 CCCGCGGATC GGTGCTGGTG CAGCGCGAGAAGGACATGTC AGCCTACA 288

From the sequence thus obtained, primers 288-41F (SEQ ID NO. 17), 288-1°F. (SEQ ID NO. 18), 288-254R (SEQ ID NO. 19) and 288-44R (SEQ ID NO. 20)were prepared and used for the 5′-RACE and 3′-RACE experiments asdescribed below. 288-41F: 5′-GGTGAACCCTGAACCCACAGGCCAACAG-3′ (SEQ ID NO.17) 288-10F: 5′-TTGGGGAGCCACTGGCAAAAATGGCACC-3′ (SEQ ID NO. 18)288-254R: 5′-TGACATGTCCTTCTCGCGCTGCACCAGC-3′ (SEQ ID NO. 19) 288-44R:5′-GGACTGTTGGCCTGTGGGTTCAGGGTTC-3′ (SEQ ID NO. 20)

5′-RACE and 3′-RACE reactions were carried out using rat liver cDNA as atemplate. The reaction solution for the PCR for 5′-RACE and 3′-RACE wasprepared by mixing 0.5 μl of Taq polymerase (TAKARA SHUZO), 5 μl of10×PCR buffer attached (500 mM KCl, 25 mM MgCl₂, 100 mM Tris.HCl, pH8.3), 4 μl of 2.5 mM dNTP mixture, 3 μl of 25 mM MgCl₂, 2 μl of 25% DMSOsolution (for 3′-RACE), 1 μl of 10 μM primer 288-10F (for 3′-RACE) or 10μM primer 288-254R (for 5′-RACE), 1 μl of 10 μM primer AP1 (primer AP1was included in the Marathon-Ready cDNA Kit supplied by CLONTECH), 5 μlof template rat liver cDNA (Marathon-Ready cDNA Kit, rat liver,CLONTECH) and 28.5 μl (for 3′-RACE) or 30.5 μl (for 5′-RACE) ofdistilled water. The conditions for the reaction were as follows: 94° C.for 60 seconds for initial denaturation, followed by 5 cycles of 94° C.for 30 seconds and 72° C. for 120 seconds, 5 cycles of 94° C. for 30seconds and 70° C. for 120 seconds, 25 cycles of 94° C. for 20 secondsand 68° C. for 120 seconds and final extension at 68° C. for 10 minutes.

Subsequently, nested PCR was performed using the PCR product solution asa template. The reaction solution was prepared by mixing 0.5 μl of Taqpolymerase (TAKARA SHUZO), 5 μl of 10×PCR buffer (500 mM KCl, 25 mMMgCl₂, 100 mM Tris.HCl, pH 8.3), 4 μl of 2.5 mM dNTP mixture, 3 μl of 25mM MgCl₂, 2 μl of 25% DMSO solution (for 3′-RACE), 1 μl of 10 μM primer288-41F (for 3′-RACE) or 10 μM primer 288-44R (for 5′-RACE), 1 μl of 10μM primer AP2 (primer AP2 was included in the Marathon-Ready cDNA Kitsupplied by CLONTECH), 5 μl of template DNA (50-fold dilution of the PCRproduct) and 28.5 μl (for 3′-RACE) or 30.5 μl (for 5′-RACE) of distilledwater. The reaction conditions were as follows: initial denaturation at94° C. for 60 seconds, followed by 5 cycles of 94° C. for 30 seconds and72° C. for 120 seconds, 5 cycles of 94° C. for 30 seconds and 70° C. for120 seconds, 25 cycles of 94° C. for 20 seconds and 68° C. for 120seconds and final extension at 68° C. for 10 minutes.

From the nested PCR product, a DNA fragment was recovered according tothe method as described above. The DNA fragment were ligated to plasmidvector pCR2.1 using the TOPO-TA Cloning Kit (Invitrogen) in accordancewith the manufacturer's instruction, and used to transform E. coliTOP10. E. coli was picked up from a single colony and liquid-cultured inLB medium. After the culture, the cell was collected and the plasmid waspurified using a plasmid purification kit (QIAwell 8 Ultra plasmidpurification kit: QIAGEN). The nucleotide sequence of the PCR productinserted into the plasmid was analyzed according to the method asdescribed above to obtain sequence information of the 5′- and 3′ ends.Based on this information, primers rKiSS364F and rKiSS859R wereprepared. (SEQ ID NO. 21) rKiSS364F:5′-CGTCTCAGCCTCTGGACACCCTGTGGATCTGCC-3′ (SEQ ID NO. 22) rKiSS859R:5′-TGGCGACAGCATTGCTTTTATTGCACAAGTCTA-3′

PCR was performed with primers rKiSS364F and rKiSS859R, using rat livercDNA as a template. The reaction solution for the PCR was prepared bymixing 1 μl of Pfu DNA polymerase (Stratagene), 5 μl of 10×PCR buffer(500 mM KCl, 25 mM MgCl₂, 100 mM Tris.HCl, pH 8.3), 4 μl of 2.5 mM dNTPmixture, 2 μl of 25% DMSO solution, 1 μl each of 10 μM primers rKiSS364Fand rKiSS859R, 5 μl of template rat liver cDNA (Marathon-Ready cDNA Kit,rat liver, CLONTECH) and 31 μl of distilled water. The conditions forthe reaction were as follows: initial denaturation at 94° C. for 60seconds, followed by 3 cycles of 94° C. for 20 seconds and 72° C. for120 seconds, 3 cycles of 94° C. for 20 seconds and 70° C. for 120seconds, 3 cycles of 94° C. for 20 seconds and 68° C. for 120 seconds,30 cycles of 94° C. for 20 seconds, 63° C. for 30 seconds and 68° C. for120 seconds, and final extension at 68° C. for 10 minutes. The DNAfragment thus obtained was cloned into pPCR-BluntII-TOPO vector(Stratagene) according to the method provided in the manufacturer'sinstruction. The cloned DNA sequence was analyzed according to themethod as described above, and pRKISS4, which contains a 393 bp DNAfragment encoding the complete peptide of rat KiSS-1, was successfullyobtained. E. coli TOP10 transformed with the plasmid was designatedTOP10/pRKISS4.

The amino acid sequence of the rat KiSS-1 protein (SEQ ID NO. 6) deducedfrom the nucleotide sequence obtained showed high homology with itsknown human homolog (see FIG. 1).

pRKISS4(393 bp): (SEQ ID NO. 23) ATGACCTCGC TGGCTTCTTG GCAGCTGCTGCTTCTCCTCT GTGTGGCCTC TTTTGGGGAG  60 CCACTGGCAA AAATGGCACC TGTGGTGAACCCTGAACCCA CAGGCCAACA GTCCGGACCC 120 CAGGAACTCG TTAATGCCTG GCAAAAGGGCCCGCGGTATG CAGAGAGCAA GCCTGGGGCT 180 GCAGGACTGC GCGCTCGCCG AACATCGCCATGCCCGCCGG TGGAGAACCC CACGGGGCAC 240 CAGCGGCCCC CGTGTGCCAC CCGCAGTCGCCTGATCCCTG CGCCCCGCGG ATCGGTGCTG 300 GTGCAGCGCG AGAAGGACAT GTCAGCCTACAACTGGAACT CCTTTGGCCT GCGCTACGGC 360 AGGAGGCAGG TGGCGCGGGC GGCACGGGGCTGA 393

EXAMPLE 3

Cloning and Sequencing of cDNA Encoding a Novel G-Protein CoupledReceptor Protein Derived from Mouse Whole Brain

PCR was performed using mouse whole brain cDNA (CLONTECH) as a templateand two primers: primer 1 (SEQ ID NO. 26) and primer 2 (SEQ ID NO. 27).The composition of the reaction solution used in the reaction was asfollows: 1/10 volume of the above cDNA was used as a template, and mixedwith 1/50 volume of Pfu Turbo DNA Polymerase (STRATAGENE), 0.2 μM eachof primer 1 and primer 2, 200 μM dNTPs, and the buffer attached to theenzyme product. The volume of the reaction solution was adjusted to 25μl. The PCR reaction was carried out as follows: (i) 94° C. for 2minutes, followed by (ii) 3 cycles of 94° C. for 20 seconds and 72° C.for 2 minutes, (iii) 3 cycles of 94° C. for 20 seconds and 68° C. for 2minutes, (iv) 38 cycles of 94° C. for 20 seconds, 62° C. for 20 secondsand 68° C. for 1 minute and 30 seconds, and final extension at 68° C.for 7 minutes. The product obtained from the PCR were subcloned into aplasmid vector pCR-Blunt II-TOPO (Invitrogen), according to themanufacturer's instruction for the Zero-blunt TOPO-TA Cloning Kit(Invitrogen). The plasmid was then introduced into E. coli DH5a andclones harboring the cDNA were selected on LB-agar medium containingkanamycin. As the result of analyzing the sequence of each clone, a cDNAsequence encoding a novel G-protein coupled receptor protein (SEQ ID NO.25) was obtained. The amino acid sequence deduced from the nucleotidesequence consisted of 396 residues (SEQ ID NO. 24) and showed thehighest homology of 94.4% with a known G-protein coupled receptorprotein rOT7T175 (GPR54) and 82.4% homology with its human counterpart,hOT7T175 (WO 00/24890). Therefore, the amino acid sequence wasconsidered to be a mouse counterpart of these proteins. Thus, the novelG-protein coupled receptor protein comprising the amino acid sequencewas designated mOT7T175. The transformant harboring the sequenceencoding mOT7T175 was designated Escherichia coli DH5a/pCR-BlundII-mOT7T175.

INDUSTRIAL APPLICABILITY

The present invention provides novel ligand proteins derived from ratsand mice and partial peptides thereof, or salts thereof, andpolynucleotides (e.g. DNA, RNA, and derivatives thereof) encoding them.Also provided are vectors containing any of said polynucleotides, amethod for producing said ligand proteins using any of said vectors.Further provided are antibodies and antagonists against said proteinsand the like, and a method and kit of screening for them.Pharmaceuticals and the like that comprise any of said proteins,polynucleotides or antibodies or antagonist against them are alsoprovided.

1. A protein comprising the same or substantially the same amino acidsequence as that represented by SEQ ID NO:1 or NO:3, or a salt thereof.2. The protein or salt thereof according to claim 1, whereinsubstantially the same amino acid sequence is the sequence representedby SEQ ID NO:2.
 3. A partial peptide of the protein according to claim1, which comprises the amino acid sequence of from the 132 to 141thresidues from the N terminal of the amino acid sequence represented bySEQ ID NO:1, or a salt thereof.
 4. The partial peptide or salt thereofaccording to claim 3, which comprises the same or substantially the sameamino acid sequence as that of from the 127 to 141th residues from the Nterminal of the amino acid sequence represented by SEQ ID NO:
 1. 5 Thepartial peptide or salt thereof according to claim 3, which has the sameor substantially the same amino acid sequence as that of from the 90 to141th residues from the N terminal of the amino acid sequencerepresented by SEQ ID NO:1.
 6. A partial peptide of the proteinaccording to claim 1, which has the same or substantially the same aminoacid sequence as that of from the 94 to 145th residues from the Nterminal of the amino acid sequence represented by SEQ ID NO:2, or asalt thereof.
 7. A partial peptide of the protein according to claim 1,which comprises the amino acid sequence of from the 110 to 119thresidues from the N terminal of the amino acid sequence represented bySEQ ID NO:3, or a salt thereof.
 8. The partial peptide or salt thereofaccording to claim 7, which comprises the same or substantially the sameamino acid sequence as that of from the 105 to 119th residues from the Nterminal of the amino acid sequence represented by SEQ ID NO:3.
 9. Thepartial peptide or salt thereof according to claim 7, which has the sameor substantially the same amino acid sequence as that of from the 68 to119th residues from the N terminal of the amino acid sequencerepresented by SEQ ID NO:3.
 10. A polynucleotide comprising apolynucleotide encoding the protein according to claim
 1. 11. Apolynucleotide comprising a polynucleotide encoding the partial peptideaccording to claim 3, 6 or
 7. 12. The polynucleotide according to claim10 or 11, which is DNA.
 13. The polynucleotide according to claim 10,which has the nucleic acid sequence represented by SEQ ID NO:4, NO:5 orNO:6.
 14. A recombinant vector comprising the polynucleotide accordingto claim 10 or
 11. 15. A transformant transformed with the recombinantvector according to claim
 14. 16. A method of producing the protein orsalt thereof according to claim 1, or the partial peptide or saltthereof according to claim 3, 6 or 7, which comprises culturing thetransformant according to claim 15, and making it produce and accumulatethe protein according to claim 1, or the partial peptide according toclaim 3, 6 or
 7. 17. An antibody to the protein or salt thereofaccording to claim 1, or the partial peptide or salt thereof accordingto claim 3, 6 or
 7. 18. The antibody according to claim 17, which is aneutralizing antibody capable of inactivating signal transduction of theprotein according to claim 1 or the partial peptide according to claim3, 6 or
 7. 19. A method of screening a compound or salt thereof whichalters the binding property between a receptor and the protein or saltthereof according to claim 1 or the partial peptide or salt thereofaccording to claim 3, 6 or 7, which comprises using the protein or saltthereof according to claim 1 or the partial peptide or salt thereofaccording to claim 3, 6 or
 7. 20. A kit for screening a compound or saltthereof which alters the binding property between a receptor and theprotein or salt thereof according to claim 1 or the partial peptide orsalt thereof according to claim 3, 6 or 7, which comprises the proteinor salt thereof according to claim 1 or the partial peptide or saltthereof according to claim 3, 6 or
 7. 21. The screening method accordingto claim 19 or the screening kit according to claim 20, wherein thereceptor is a protein or salt thereof having the same or substantiallythe same amino acid sequence as that represented by SEQ ID NO:7, NO:8 orNO:24.
 22. A compound or salt thereof which alters the binding propertybetween a receptor and the protein or salt thereof according to any oneof claims 1, 3, 4 and 5, which is obtainable using the screening methodaccording to claim 19 or the screening kit according to claim
 20. 23.The compound or salt thereof according to claim 22, which is an agonist.24. A pharmaceutical composition comprising a compound or salt thereofwhich alters the binding property between a receptor and the protein orsalt thereof according to claim 1 or the partial peptide or salt thereofaccording to claim 3, 6 or 7, which is obtainable using the screeningmethod according to claim 19 or the screening kit according to claim 20.25. The pharmaceutical composition according to claim 24, which is anagent for inhibiting tumor metastasis.
 26. A method of quantifying theprotein according to claim 1 or the partial peptide according to claim3, 6 or 7, which comprises using the antibody according to claim
 17. 27.A pharmaceutical composition comprising the protein or salt thereofaccording to claim 1 or the partial peptide or salt thereof according toclaim 3, 6 or
 7. 28. The pharmaceutical composition according to claim27, which is an agent for inhibiting tumor metastasis.